CN110144353B - A rubber tree U6 gene promoter proHbU6.6 and its cloning and application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, in particular to a rubber tree RNA polymerase type III promoter, more specifically to a rubber tree U6 gene promoter proHbU6.6, and further discloses its cloning method and application. The present invention clones the rubber tree RNA polymerase III type promoter in Hevea brasiliensis for the first time - the rubber tree endogenous U6 promoter proHbU6.6, the promoter is the rubber tree endogenous RNA polymerase type III promoter, and the promoter has efficient transcription The activity of the promoter can drive the expression of downstream sgRNA, and the activity of the promoter and its feasibility in the rubber tree CRISPR/Cas9 gene editing system were verified by transient transformation of rubber tree protoplasts, and the CRISPR/Cas9-mediated rubber tree genome target was realized to the editor.

Description

A rubber tree U6 gene promoter proHbU6.6 and its cloning and application

technical field

The invention belongs to the technical field of genetic engineering, in particular to a rubber tree RNA polymerase type III promoter, more specifically to a rubber tree U6 gene promoter proHbU6.6, and further discloses its cloning method and application.

Background technique

Natural rubber has always been an important strategic material and reserve resource in my country. At present, the selection and breeding of new rubber tree varieties in my country is still based on traditional cross-breeding. Due to the slow growth of rubber trees, conventional breeding has problems of low efficiency and long cycle, which seriously hinders the progress of rubber tree breeding. With the development of biotechnology, the application of molecular breeding technology in rubber trees has accelerated the cultivation of new varieties of rubber trees, and the introduction of exogenous genes through Agrobacterium or gene gun has become an important way of genetic improvement of rubber trees. However, in the process of the above-mentioned introduction of exogenous gene fragments, the gene fragments are randomly inserted into the rubber tree genome, not only the insertion site is not controlled, but also there are problems such as location benefits (Yutaka et al., 2018, Chromosoma, doi: 10.1007/ s00412-018-0677-6.).

The emergence of CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/CRISPR-associated nuclease 9) genome editing technology provides a new approach for precise genome modification (Yongwei et al., 2016, Front Plant Sci, 7:1928; Collonnier et al., 2017, Methods, 121-122: 103-117; Yutaka et al., 2018, Chromosoma, doi: 10.1007/s00412-018-0677-6), the basic principle of which mediates gene editing is to rely on single-stranded guide RNA (single guide RNA, sgRNA) ), the nuclease Cas9 is used to site-specifically bind and cleave specific target sites upstream of the genome-specific site (PAM), resulting in DNA double-strand breaks (DSBs), which in turn cause mutation of the target site. At the same time, through homologous recombination repair (HR) or non-homologous end joining (NHEJ) biological endogenous repair mechanisms, exogenous donor DNA can also be inserted into the sgRNA target site, so as to achieve mutation of specific sites on the genome And the knockout, insertion and replacement of specific DNA fragments (Lee et al., 2017, Elife, 6: e25312.; Smirnikhina et al., 2018, Hum Genet, 138(1): 1-19.), and thus achieve the target gene precise variety improvement. Currently, in Arabidopsis (Miki D et al., 2018, Nat Commun. 9(1): 1967.), rice (Li J et al., 2016, Nat Plants, 2(10): 16139.) and maize (Gil-Humanes J et al, 2017, Plant J, 89(6): 1251-1262.) and other plants have used plasmid-based CRISRP/Cas9 genome editing technology to achieve the introduction of foreign genes. In addition, the introduction of foreign genes has also been achieved by CRISPR/Cas9-sgRNA RNP transient transformation in maize, wheat, and rice (Svitashev S et al., 2015, Plant Physiol, 169(2): 931-945.; Liang Z et al., 2017, Nat Commun, 8:14261.; Sun Y W et al., 2016, Mol Plant, 9(4):628-631); in addition, Liang et al. (2018, Nature Protocols, 13(3):413-430.) Gene editing was also achieved by transiently transforming recipient cells with Cas9 and sgRNA expression vector plasmids using a gene gun.

Studies have shown that in the CRISPR/Cas9 gene editing system, the content level of sgRNA in recipient cells is one of the important factors affecting editing efficiency. Therefore, polymerase type III promoters that can precisely initiate sgRNA transcription in vivo have received extensive attention. (Jinek et al, 2016, Science 337:816-821; Mali et al, 2013, Science 339:823-826; Cong et al, 2013, Science 339:819-823). U6 RNA is a non-coding RNA involved in pre-mRNA splicing, and its corresponding U6 promoter is a class of RNA polymerase type III promoters, and has been widely used in CRISPR/Cas9 systems in many species (Kim and Nam,, 2013, plant mol.Bio.Rep.31:581-593; Li et al., 2007, J.integrate plant biol..49:222-229; H.Jia et al., 2014, pone, 9(4): e93806.).

Although CRISPR/Cas9 genome editing technology has been widely used in many species, the gene editing technology for rubber tree has not been reported. This is mainly due to the fact that although U6 promoters have been extensively reported in many species, exogenous U6 promoters are generally not applicable (X. Sun et al., 2015, Sci. Rep., 5p. 10342). It can be seen that the lack of a suitable U6 promoter has become a limiting factor for the current rubber tree CRISPR/Cas9 gene editing system, and also limits the application of CRISPR/Cas9 genome editing technology in rubber tree breeding. Therefore, screening out the functionally active U6 promoter in rubber tree has positive significance for the development of genetic breeding technology of rubber tree.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is to provide a rubber tree U6 gene promoter proHbU6.6, and further disclose its cloning method and application.

In order to solve the above-mentioned technical problems, a rubber tree U6 gene promoter proHbU6.6 according to the present invention, the promoter proHbU6.6 includes the DNA nucleotide sequence shown in SEQ ID No: 1. The above-mentioned Hevea U6 gene promoter proHbU6.6 belongs to the RNA polymerase III type promoter of the Hevea U6 snRNA gene, and is derived from Heveabrasiliensis.

Specifically, the DNA nucleotide sequence of the promoter proHbU6.6 is shown in SEQ ID No: 1.

The invention also discloses a rubber tree transient transformation editing vector, which contains the rubber tree U6 gene promoter proHbU6.6.

Specifically, the transient transformation editing vector is a recombinant plasmid proHbU6.6-sgRNA-163Cas9M.

The present invention also discloses a method for cloning described rubber tree U6 gene promoter proHbU6.6, comprising the following steps:

(1) The following specific primers were designed with the genomic DNA of the Hexa brasiliensis 7-33-97 leaves as the template:

proHbU6.6-F: ATCTAACATTGTCTTGCTTC;

proHbU6.6-R:CGGTTGCTCAATGCTTCGTG;

(2) PCR amplification was performed in a 20 μl reaction system using KOD FX enzyme;

(3) The amplified product TA was cloned into the pMD19-T vector, transformed into Escherichia coli Dh5α, and the recombinant vector was single-cloned for sequencing to obtain the 462bp rubber tree U6 gene promoter DNA fragment proHbU6.6.

Specifically, in the step (2), the reaction procedure of the PCR amplification step is: pre-denaturation at 95°C for 2 min, denaturation at 98°C for 10s, annealing at 59°C for 30s, extension at 72°C for 1 min, 35 cycles, and finally at 72°C Extend 5min.

The present invention also discloses a method for constructing the instant transformation and editing vector of rubber tree, comprising the following steps:

(a) Construction of the cloned promoter proHbU6.6 into the intermediate vector SK-5G

The SK-5G vector was digested with SalI and XhoI, and the 2913bp vector backbone fragment was recovered. The following primers were designed to introduce homologous sequences at both ends of the proHbU6.6 sequence and the vector gRNA sequence, respectively. ProHbU6.6 and gRNA were assembled into the SK-5G vector Get proHbU6.6-SK-5G on:

proHbU6.6-lF:

GCGGCCGCAGATCTGCTAGCGTCGAC ATCTAACATTGTCTTGCTTC;

proHbU6.6-lR:

GGTGTTGTGTTCACCTGCGAGCCGGTTGCTCAATGCTTCGTG ;

gRNA-sF: GCTCGCAGGTGAACACAACACC;

gRNA-sR: TTGGGTACCGAGGATCCTCTAGA;

(b) Construction of the target site into the proHbU6.6-SK-5G vector

The following target sites were selected on the rubber tree HbTFL1-3 gene:

GCCCAGCATAGGGATCCAC, the proHbU6.6-SK-5G vector was digested with AarI to form two sticky ends of 3'-TGGC and 5'-GTTT;

Synthesize the target site sequence and introduce two sticky ends, 5'-ACCG and 3'-CAAA (Figure 3):

Forward: ACCGGCCCCAGCATAGGGATCCAC;

Reverse: AAACGTGGATCCCTATGCTGGGC;

The above-mentioned forward and reverse target sequence DNAs were mixed, treated at 100°C, and then annealed at room temperature to form double-stranded DNA with cohesive ends complementary to the proHbU6.6-SK-5G vector, which was then ligated with T4 DNA ligase. The fragment is connected to the downstream position of the proHbU6.6 promoter of the vector to obtain a complete sgRNA expression cassette;

(c) Construction of sgRNA expression cassette into transient transformation editing vector 163Cas9M

The proHbU6.6-sgRNA-SK-5G vector was digested with KpnI and BglII, and a small fragment of 625bp sgRNA expression cassette was recovered;

The 163Cas9M vector was double digested with KpnI and BamHI, and a large fragment of 7919bp was recovered;

Use T4 DNA ligase to construct the small fragment sgRNA expression cassette into the 163Cas9M vector to obtain the rubber tree transient transformation editing vector proHbU6.6-sgRNA-163Cas9M.

The invention also discloses a method for genome editing of rubber tree, which comprises the step of introducing the rubber tree transient transformation editing vector into rubber tree protoplast.

The invention also discloses the application of the rubber tree U6 gene promoter proHbU6.6 in the technical field of rubber tree molecular breeding.

The invention also discloses the application of the rubber tree transient transformation editing vector in the technical field of rubber tree molecular breeding.

The present invention clones the rubber tree RNA polymerase III type promoter in Hevea brasiliensis for the first time-the rubber tree endogenous U6 promoter proHbU6.6, the promoter is the rubber tree endogenous RNA polymerase type III promoter, and the promoter has efficient transcription It can drive the expression of downstream sgRNA, and after screening the target sequence of the endogenous flowering regulatory gene HbTFL1-3 in rubber tree, it was constructed into the editing vector downstream of the proHbU6.6 promoter, and PEG-mediated transient transformation of rubber tree leaf protoplasm The editing vector was introduced into the rubber tree cells to realize the editing of the HbTFL1-3 gene target site, and the activity of the proHbU6.6 promoter was verified. For the first time, an effective CRISPR/Cas9 gene editing technology system was established in rubber tree.

In the present invention, the cloned rubber tree endogenous RNA polymerase type III promoter is used for the first time to transform rubber tree protoplasts to verify the activity of the promoter and the feasibility of applying it to the rubber tree CRISPR/Cas9 gene editing system, and realize the CRISPR/Cas9 gene editing system for the first time. /Cas9-mediated targeted editing of the rubber tree genome. It has been verified that the results of recombinant cloning and sequencing of the edited target gene fragments show that most of the mutations are single-base deletions, including single-base insertions and small fragment deletions. . Therefore, the cloned promoter proHbU6.6 of the present invention can be applied to the rubber tree CRISPR/Cas9 gene editing system, thereby realizing efficient and accurate variety improvement of rubber tree.

Description of drawings

In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below according to specific embodiments of the present invention and in conjunction with the accompanying drawings, wherein,

Fig. 1 shows the sequence alignment of HbU6.6 gene of rubber tree with Arabidopsis thaliana and cotton U6 gene, wherein, the position of the box line is the key element USE (upstream sequence element) and TATA box of U6 snRNA transcription, and the position of the arrow is the transcription initiation site ;

Fig. 2 is the cloning electrophoresis diagram of the HbU6.6 gene promoter of rubber tree, it can be seen that the HbU6.6 gene promoter fragment of 462bp has been obtained by PCR amplification;

Figure 3 is a schematic structural diagram of the rubber tree transient transformation editing vector proHbU6.6-sgRNA-163Cas9M, wherein the target sequence is in the frame, the underlined sequence is the BamHI recognition enzyme cleavage site, and its two ends are the cohesive ends complementary to the vector;

Figure 4 is the identification result of PCR product re-enzyme digestion (PCR/RE) of the target gene fragment of rubber tree protoplast genome; wherein, 1: PCR product of rubber tree protoplast DNA target gene fragment; 2: protoplast DNA target gene fragment transformed into control 163Cas9M vector The PCR product was then digested with BamHI; 3: The PCR product of the protoplast DNA target gene fragment of proHbU6.6-sgRNA-163Cas9M vector was digested with BamHI; the unedited target gene fragment was digested into 500bp and 331bp small fragments by BamHI, The target gene fragment after gene editing mutation is the band at the arrow that cannot be cut by enzyme;

Figure 5 is a graph showing the result of enzyme digestion verification of the target gene fragment in the recombinant clone after partial TA cloning. The single clone of the target gene fragment recombinant vector that cannot be digested by BamHI can be obtained by screening the target gene fragment;

Figure 6 shows the sequence alignment analysis of the editing result of the target site; it can be seen that the edited target site is sequenced and compared with the wild type, and the main mutation type is single-base deletion, among which, single-base deletions also appear. Insertions and deletions of small fragments. .

Detailed ways

In the following examples of the present invention, unless otherwise specified, they are all conventional methods. The carrier SK-5G in the following embodiment and the transient transformation vector 163Cas9M are donated by the Wang Kejian research group of China Rice Research Institute (Wang Kejian, Wang Chun, Shen Lan, etc., the construction and application of the plant multigene knockout vector), and this biological material is only for It is used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

The acquisition of embodiment 1 Hevea brasiliensis U6 gene promoter proHbU6.6

With reference to the DNA sequences of the Arabidopsis AtU6-26 gene (Genebank accession number: X52528.1) and cotton GhU6-9 gene (Genebank accession number: XR_001680717.1), we searched the rubber tree genome database (hevea.catas. cn), found a rubber tree HbU6 gene (Genebank accession number: XR_002491077.1) by homologous alignment, and obtained the upstream reference sequence of this gene.

The following specific primers for proHbU6.6-F and proHbU6.6-R were designed to clone the 462bp DNA in the promoter region using the genomic DNA of the leaves of Hexa brasiliensis Reyan 7-33-97 (cultivated by the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences) as the template. Fragment:

proHbU6.6-F: ATCTAACATTGTCTTGCTTC;

proHbU6.6-R:CGGTTGCTCAATGCTTCGTG;

PCR amplification was performed using KOD FX enzyme (TOYOBO) in a 20 μl reaction system. The specific reaction procedure was: 95°C pre-denaturation for 2 min, 98°C denaturation for 10s, 59°C annealing for 30s, 72°C extension for 1 min, 35 cycles, and 72°C final Extend 5min.

The amplified product TA was cloned into the pMD19-T vector, transformed into Escherichia coli Dh5α, and the recombinant vector was picked for single clone PCR electrophoresis to verify and sequenced. The electrophoresis results are shown in Figure 2. Finally, the 462bp DNA fragment proHbU6.6 of the rubber tree U6 gene promoter shown in SEQ ID No: 1 was obtained.

Using Vector NTI align X (Invitrogen), the promoter sequence was compared with the base sequences of Arabidopsis AtU6-26 and cotton GhU6-9 and their promoters, and it was found that the promoter sequence also contained the key position of U6 snRNA transcription. TATA box and USE (upstream sequence element) elements (as shown in Figure 1), and the positions of these two elements relative to the transcription start site are also basically the same as the promoter sequences of AtU6-26 and cotton GhU6-9, This is important for the binding of geometrically symmetrical RNA polymerases (Kim and Nam, (2013) plant mol. Bio. Rep. 31:581-593).

Example 2 Construction of rubber tree gene editing vector

(1) Construction of proHbU6.6 on the intermediate vector SK-5G vector

The SK-5G vector and downstream gRNA fragments were digested with SalI and XhoI double enzymes, the rice U3 promoter on the vector was removed, the 2913bp vector backbone fragment was recovered, and primers were designed:

proHbU6.6-lF:

GCGGCCGCAGATCTGCTAGCGTCGAC ATCTAACATTGTCTTGCTTC;

proHbU6.6-lR:

GGTGTTGTGTTCACCTGCGAGCCGGTTGCTCAATGCTTCGTG ;

(underlined sequence homologous to SK-5G vector).

Using the rubber tree genomic DNA as the template, KOD FX enzyme (TOYOBO) was used in a 20 μl reaction system for pre-denaturation at 95 °C for 2 min, denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and final extension at 72 °C for 5 min. The proHbU6.6 promoter fragment was added, and the homologous sequences of SK-5G vector were introduced at both ends.

Design primers:

gRNA-sF: GCTCGCAGGTGAACACAACACC;

gRNA-sR: TTGGGTACCGAGGATCCTCTAGA;

Using the SK-5G vector plasmid as a template, KOD FX enzyme (TOYOBO) was used in a 20 μl reaction system for pre-denaturation at 95 °C for 2 min, denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and final extension at 72 °C 5min amplification to obtain gRNA fragments.

Using Gibson assembly Cloning Kit (NEB), referring to the instructions, the obtained homologous sequence proHbU6.6 promoter fragment and gRNA fragment were assembled into SK-5G vector to obtain proHbU6.6-SK-5G.

(2) Construction of the target site into the proHbU6.6-SK-5G vector

In order to verify the functional activity of the proHbU6.6 promoter, the rubber tree flowering regulatory gene HbTFL1-3 was selected as the target gene for CRISRP/Cas9 editing, and the appropriate target site GCCCAGCATAG GGATCC AC was selected on this gene, which is upstream of the PAM site AGG The 2 base positions contain the BamHI recognition site GGATCC (underlined), which can be used for restriction enzyme identification after the target site is edited.

The synthetic target site sequence is as follows:

Forward: ACCG GCCCAGCATAGGGATCCAC;

Reverse: AAAC GTGGATCCCTATGCTGGGC, two sticky ends of 5'-ACCG and 3'-CAAA are underlined.

After the proHbU6.6-SK-5G vector was digested with AarI, two sticky ends of 3'-TGGC and 5'-GTTT were formed downstream of the proHbU6.6 promoter. At the same time, 10 μl of the forward and reverse target sequences at a concentration of 100 μM were mixed, and treated at 100 °C for 5 min, and then annealed at room temperature to form a target site double-stranded DNA with cohesive ends complementary to the proHbU6.6-SK-5G vector. Fragment. Then, T4 DNA ligase (NEB) was used to ligate the fragment into the proHbU6.6-SK-5G vector at the downstream position of the proHbU6.6 promoter to obtain a complete sgRNA expression cassette (the results are shown in Figure 3).

(3) Construction of sgRNA expression cassette into transient transformation CRISRP/Cas9 gene editing vector 163Cas9M

The proHbU6.6-sgRNA-SK-5G vector was digested with KpnI and BglII, and the 625bp small fragment proHbU6.6-sgRNA expression cassette was recovered;

The 163Cas9M vector was double digested with KpnI and BamHI, and a large fragment of 7919bp was recovered;

According to the characteristics of the same sticky ends produced by BamHI and BglII digestion, T4 DNA ligase (NEB) was used to construct the small fragment sgRNA expression cassette into the 163Cas9M vector to obtain the rubber tree transient transformation editing vector proHbU6.6-sgRNA-163Cas9M (the construction results are as follows: shown in Figure 3).

Example 3 Rubber tree protoplast transformation

In this example, the PEG-mediated editing vector proHbU6.6-sgRNA-163Cas9M was used to transform rubber tree protoplasts, and the 163Cas9M vector without the sgRNA expression cassette was used as a control.

Transfer the rubber tree heat research 7-33-97 tissue culture seedlings that have been cultivated for one month to 26-28 ℃ dark conditions for 5-7 days, take 2g of leaves in the discoloration stage and soak them in 0.6M mannitol solution for 10min immediately for preparation Protoplasts. The preparation of protoplasts and the transformation process refer to (Yoo, S.D. et al., 2007, Nature Protocols, 2: 1565-1575.). The transformed protoplasts were cultured in the dark at 26-28°C for 48 hours for detection of target site mutations.

Example 4 Detection of the mutation site of the HbTFL1-3 target sequence of rubber tree

The plant genomic DNA extraction kit (Tiangen) was used to extract the genomic DNA of rubber tree protoplasts, and the PCR/Restriction Enzyme digestion (PCR/RE) method of genomic DNA was used to detect the target site sequence of HbTFL1-3 gene. Mutation (Henao-Mejia J et al., 2016, Cold Spring Harbor Protocols, 2016(2): pdb.prot090704.).

Design the PCR amplification primers for the target gene fragment:

HbTFL1-3-F: CACCTAGGGCATAACTTCTAC,

HbTFL1-3-R:ACGGGATCTTAGTTGGATGG,

Using rubber tree protoplast genomic DNA as the template, the reaction program is: 95°C pre-denaturation for 2 min, 98°C denaturation for 10s, 59°C annealing for 30s, 72°C extension for 1 min, 35 cycles, 72°C final extension for 5 min, PCR amplification to obtain the edited The 831bpHbTFL1-3 target gene fragment within the site.

Because the target gene editing site contains a BamHI restriction endonuclease recognition site, the editing of the target site was detected by BamHI digestion of the recovered HbTFL1-3 gene fragment. The results are shown in Figure 4. Some target gene fragments are no longer recognized and digested by BamHI due to the mutation of the BamHI recognition site, which preliminarily indicates that the target gene has been successfully edited, while the unedited target gene fragments are digested by the enzyme. Two small fragments of 500bp and 331bp. In order to eliminate the influence of some unedited target gene fragments remaining due to incomplete enzyme digestion, the fragments that could not be digested by the PCR products were recovered, TA cloned into the pMD-19T (TAKARA) vector, transformed into Escherichia coli Dh5α, and then again. The target gene fragment on the single clone of the recombinant vector was verified by BamHI digestion, and the results are shown in Figure 5; DNA sequencing was performed on the single clone that could not be cut by the enzyme, and the mutation of the HbTFL1-3 target site sequence of rubber tree was analyzed by sequence comparison. The editing results in rubber tree protoplast cells were confirmed. The results are shown in Figure 6.

It can be seen that the present invention obtains the rubber tree RNA polymerase type III promoter proHbU6.6 promoter in Hevea brasiliensis with transcriptional activity, which can drive the expression of downstream sgRNA, and realizes the targeted editing of the rubber tree genome mediated by CRISPR/Cas9 for the first time; Cloning and sequencing of the target site after editing revealed that most of the mutations were single-base deletions, including single-base insertions and small fragment deletions. Therefore, the promoter of the present invention can be applied to the rubber tree CRISPR/Cas9 gene editing system, thereby realizing efficient and accurate variety improvement of rubber tree.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

sequence listing

<110>

<120> A kind of rubber tree U6 gene promoter proHbU6.6 and its cloning and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 462

<212> DNA

<213> proHbU6.6

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atctaacatt gtcttgcttc ctttttttct tcttcttttt tttttaattg catgaaaatg 60

cattgcacgg ccaaaggcag attacaaaca agaatacaaa gcttctacat tggcatttct 120

gctaacgatt gaaaaagaaa tttgatccgg aatgtagctc tcgaaaatag ctctatttct 180

taccttccat atagaccaaa tggttactgt cactatgaat ttgcaattta gaatttacaa 240

gggcaaatgt ggctaaaatt tgggcctcat gatctaaaat agtaatgggt tgtagtccat 300

ggagagttta aaagttgctt gcttggtgcg tcctagtcaa tgagctgacg tgtttaaaat 360

gaatgatgac gcttaatggt tagaagagaa ccacatgccc acatcgccta gttacatgca 420

tttactagac tttatgcctg tccacgaagc attgagcaac cg 462

Claims (9)

1. A rubber tree U6 gene promoter proHbU6.6 is characterized in that, the DNA nucleotide sequence of described promoter proHbU6.6 is as shown in SEQ ID No: 1. 2. A rubber tree transient transformation editing vector is characterized in that, contains the rubber tree U6 gene promoter proHbU6.6 of claim 1. 3. The transient transformation editing vector of rubber tree according to claim 2, wherein the transient transformation editing vector is a recombinant plasmid proHbU6.6-sgRNA-163Cas9M. 4. a method for cloning the described rubber tree U6 gene promoter proHbU6.6 of claim 1, is characterized in that, comprises the steps: (1) The following specific primers were designed with the genomic DNA of the Hexa brasiliensis 7-33-97 leaves as the template: proHbU6.6-F: ATCTAACATTGTCTTGCTTC; proHbU6.6-R:CGGTTGCTCAATGCTTCGTG; (2) PCR amplification was performed in a 20 μl reaction system using KOD FX enzyme; (3) The amplified product TA was cloned into the pMD19-T vector, transformed into Escherichia coli Dh5α, and the recombinant single clone was sequenced to obtain a 462 bp rubber tree U6 gene promoter DNA fragment proHbU6.6. 5. the method for the described rubber tree U6 gene promoter proHbU6.6 of cloning according to claim 4, is characterized in that, in described step (2), the reaction program of described PCR amplification step is: 95 ℃ of preliminary Denaturation for 2 min, denaturation at 98 °C for 10 s, annealing at 59 °C for 30 s, extension at 72 °C for 1 min, 35 cycles, and final extension at 72 °C for 5 min. 6. a method for constructing the instant transformation editing carrier of rubber tree described in claim 2 or 3, is characterized in that, comprises the steps: (a) Construction of the cloned promoter proHbU6.6 into the intermediate vector SK-5G The SK-5G vector was digested with SalI and XhoI, and the 2913bp vector backbone fragment was recovered. The following primers were designed to introduce homologous sequences at both ends of the proHbU6.6 sequence and the vector gRNA sequence, respectively. ProHbU6.6 and gRNA were assembled into the SK-5G vector Get proHbU6.6-SK-5G on: proHbU6.6-lF: GCGGCCGCAGATCTGCTAGCGTCGAC ATCTAACATTGTCTTGCTTC; proHbU6.6-lR: GGTGTTGTGTTCACCTGCGAGCCGGTTGCTCAATGCTTCGTG ; gRNA-sF: GCTCGCAGGTGAACACAACACC; gRNA-sR: TTGGGTACCGAGGATCCTCTAGA; (b) Construction of the target site into the proHbU6.6-SK-5G vector The following target sites were selected on the rubber tree HbTFL1-3 gene: GCCCAGCATAGGGATCCAC, the proHbU6.6-SK-5G vector was digested with AarI to form two sticky ends of 3'-TGGC and 5'-GTTT; Synthesize the target site sequence and introduce two sticky ends, 5'-ACCG and 3'-CAAA: Forward: ACCGGCCCCAGCATAGGGATCCAC; Reverse: AAACGTGGATCCCTATGCTGGGC; The above-mentioned forward and reverse target sequence DNAs were mixed evenly, treated at 100°C, and then annealed at room temperature to form double-stranded DNA with cohesive ends complementary to the proHbU6.6-SK-5G vector. The fragment is connected to the downstream position of the proHbU6.6 promoter of the vector to obtain a complete sgRNA expression cassette; (c) Construction of sgRNA expression cassette into transient transformation editing vector 163Cas9M The proHbU6.6-sgRNA-SK-5G vector was digested with KpnI and BglII, and a small fragment of 625bp sgRNA expression cassette was recovered; The 163Cas9M vector was double digested with KpnI and BamHI, and a large fragment of 7919bp was recovered; Use T4 DNA ligase to construct the small fragment sgRNA expression cassette into the 163Cas9M vector to obtain the rubber tree transient transformation editing vector proHbU6.6-sgRNA-163Cas9M. 7. A method for genome editing of rubber tree, characterized by comprising the step of introducing the rubber tree transient transformation editing vector described in claim 2 or 3 into rubber tree protoplasts. 8. The application of the rubber tree U6 gene promoter proHbU6.6 of claim 1 in the technical field of rubber tree molecular breeding. 9. The application of the instantaneous transformation editing vector of rubber tree described in claim 2 or 3 in the technical field of rubber tree molecular breeding.

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