CN106086063B - RNAi vector constructed based on isocaudarner and application thereof - Google Patents

Abstract

The invention discloses an RNAi vector constructed based on isocaudal enzyme, which is characterized in that the vector construction method is as follows: introducing a target fragment of a targeting gene into a transition vector, and constructing an RNAi vector capable of producing a hairpin structure RNA; The method utilizes the properties of isocaudal enzymes, and only needs to obtain a DNA fragment through one PCR amplification, and digest the fragment once, which can simplify the construction process and improve the construction efficiency. At the same time, the part of the RNAi vector constructed by this method that forms the hairpin structure (target fragment-intron-inverted repeat target fragment) only needs two isocaudal enzyme cleavage sites, which can reserve more single Cloning site to improve the flexibility of vector construction. The RNAi vector constructed by this method can be widely used in functional research, genetic modification and molecular breeding of genes of multiple species, and has very important value in basic research and agricultural production.

Description

An RNAi vector constructed based on isocaudal enzyme and its application

(1) Technical field

The present invention relates to an RNAi carrier, in particular to an RNAi carrier constructed based on isocaudal enzyme and its application.

(2) Background technology

RNA interference (RNAi) is a ubiquitous sequence-specific post-transcriptional gene silencing in animals and plants caused by homologous double-stranded RNA of target genes (Hannon, (2002) Nature, 418:244-251). It was first discovered in plants and has now become an important research method in the post-genome era. RNAi has important application value in plant functional genome, growth and development, anti-virus, quality improvement and so on.

In plants, RNAi is generally achieved by a hairpin RNA (hairpin RNA (hpRNA)) with double-stranded RNA (dsRNA) (Waterhouse and Helliwell, (2003) Nat Rev Genet, 4:29-38). Although antisense RNA-mediated gene silencing as an RNAi phenomenon has been widely used in plant gene function analysis, hpRNA-mediated RNAi has a higher efficiency (Chuang and Meyerowitz, (2000) P Natl Acad Sci USA 97:4985 -4990). The hpRNA-producing vector is usually cloned from the target gene into a set of reverse complementary nucleotide sequences, which are connected by an unrelated spacer sequence, and then driven by a strong promoter for expression. The promoter can be 35S CaMV (mostly used in dicotyledonous plants) or maize ubiquitin 1 (mostly used in monocotyledonous plants). The irrelevant spacer sequence in the middle of the reverse complement sequence is preferably an intron (Intron), which is very important for the stability of the inverted repeat sequence in Escherichia coli and the improvement of gene silencing efficiency in plants (Smith et al. ., (2000) Nature 407:319-320; Wesley et al., (2001) Plant J 27:581-590). The above-mentioned vectors for producing hpRNA are relatively high in stability and efficiency, but the construction of the vectors is relatively complicated, and a simple and efficient vector construction method for producing hpRNA needs to be discovered.

Isocaudal enzymes refer to restriction endonucleases that can cleave to produce identical cohesive ends. Commonly used isocaudal enzymes include: BglII and BamHI, NheI and XbaI, SalI and XhoI, etc. The properties of these isocaudal enzymes can be used to simplify the construction of hpRNA-producing RNAi vectors.

(3) Contents of the invention

The purpose of the present invention is to provide an RNAi vector constructed based on isocaudal enzyme and its application, and to solve the technical problem that multiple target fragments (forward sequence, reverse sequence, intron) need to be cloned to construct a hairpin RNA vector at present. This method only needs to clone a target fragment by PCR to construct an RNAi vector with high interference efficiency.

The technical scheme adopted in the present invention is:

The present invention provides an RNAi vector constructed based on isocaudal enzyme. The vector construction method is as follows: introducing a target fragment of a targeting gene into a transition vector, and constructing an RNAi vector capable of producing a hairpin structure RNA;

An intron and a terminator are preset in the transition vector, the 5' end of the intron is sequentially set with homozygous enzyme cleavage sites A1, B1, and the 3' end is sequentially set with homozygous enzyme cleavage sites B2, A2, where A1 and A2 are a group of homozygous enzyme cleavage sites, B1 and B2 are another group of homozygous enzyme cleavage sites, the letters themselves have no meaning, in order to facilitate the expression of the enzyme cleavage sites designed at both ends of the intron The points are named from two groups of homologous enzymes; the 5' end and 3' end of the target fragment are respectively set with A1, B1 or A2, B2 restriction sites; through two enzyme digestion, ligation and transformation reactions, the target fragment is The 5' end and 3' end of the intron are respectively connected to form a DNA fragment with the structure of "target fragment-intron-inverted repeat target fragment-terminator", and then the above-mentioned DNA fragments are digested, ligated and transformed by one time. The DNA fragment is ligated into the final vector with a preset promoter, or the DNA fragment and the designed promoter are ligated into the final vector through three-segment ligation to obtain an RNAi vector.

Further, the isocaudal enzyme is one of the following: (1) BglII and BamHI, (2) NheI and XbaI, (3) SalI and XhoI. Other restriction enzymes with similar properties can also be used.

Further, the intron nucleotide sequence is shown in SEQ ID NO.1.

Further, the nucleotide sequence of the terminator is shown in SEQ ID NO.2.

Further, the vector is pGEM-T easy Vector.

Further, the final vector is a binary vector pCambia1300-pZmUbi-G10.

Further, the described RNAi carrier construction method based on isocaudal enzyme construction is:

(1) Take pGEM-T easy Vector as the backbone, connect the intron and terminator into the vector, and set the A1, B1 and B2, A2 enzyme cleavage sites respectively at both ends of the intron to form a transition vector 1; (2) Enzymatic digestion and recovery of the target gene with A1, B1 or A2, B2 restriction sites at both ends obtained by PCR amplification to obtain the target fragment;

(3) The transition vector 1 is then digested and recovered with two isocaudal enzymes arranged on the target gene to obtain a vector;

(4) linking and transforming the target fragment recovered in step (2) and step (3) with the vector, to obtain a transition vector 2 containing a section of the target gene;

(5) The transition vector 2 is digested and recovered with the corresponding homocaudal enzyme provided with the same target gene restriction site, and is ligated and transformed with the target fragment recovered in step (2) to obtain a target fragment with "target fragment-internal". The transition vector 3 of the structure of "intron-inverted repeat target fragment";

(6) Finally, the "target fragment-intron-inverted repeat target fragment" structure in the transition vector 3 is recovered together with the terminator enzyme digestion and then linked into the corresponding final vector, that is, the RNAi vector is obtained.

The present invention also provides an application of the RNAi vector constructed based on the isocaudal enzyme in gene transformation. This method can be used to construct an RNAi vector that produces a hairpin structure RNA, which can be transgenic into plant or animal cells to achieve silencing of the target gene.

The present invention also provides an application of the RNAi vector constructed based on isocaudal enzyme in the preparation of transgenic plants. The RNAi vector can be constructed by this method and introduced into plant cells by transgenic method to obtain a transgenic plant whose target gene is silenced. This method can be used for the study of gene function, as well as for inhibiting and silencing the expression of target genes and improving crop traits.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

Compared with the existing RNAi vector construction methods for producing hpRNA, the method of the present invention utilizes the characteristics of isocaudal enzymes, and only needs to obtain a DNA fragment through one PCR amplification, and the fragment is digested once, which can simplify the construction process and improve the efficiency of the process. Build efficiency. At the same time, the part of the RNAi vector constructed by this method that forms a hairpin structure (target fragment-intron-inverted repeat target fragment) only needs two isocaudal enzyme cleavage sites, which can reserve more single Cloning site to improve the flexibility of vector construction. The RNAi vector constructed by this method can be widely used in functional research, genetic modification and molecular breeding of genes of multiple species, and has very important value in basic research and agricultural production.

(4) Description of drawings

Figure 1: Schematic diagram of the technical scheme for the construction of RNAi vectors based on isocaudal enzymes.

Figure 2: Schematic diagram of the pGEM-T-LOG-intron-Ter vector structure. LOG-Intron is the second intron of rice LOG gene. The 5' end of LOG-Intron is provided with 4 restriction sites, namely EcoRI, HindIII, BamHI, and XhoI, of which EcoRI and HindIII are used for final vector construction, and BamHI and XhoI are one of the two groups of isocaudal enzymes, respectively. To connect the target fragment; the 3' end is provided with SalI and BglII restriction sites in turn, and SalI and BglII are the homologous enzymes of XhoI and BamHI respectively. Between ApaI and SacI is a synthetic terminator Ter.

Figure 3: Schematic diagram of the structure of the RNA part that generates the hairpin structure in the RNAi final vector with the target fragment inserted. p35S: 35S promoter; F: the target fragment targeting the target gene; LOG-Intron: the second intron of the rice LOG gene; R: the inverted repeat sequence of the target fragment targeting the target gene; Ter: artificially synthesized terminator. After the fragment R was ligated into the vector, the restriction sites SalI and XhoI, BglII and BamHI were ligated and blunted.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Embodiment 1, RNAi transition vector construction

Obtaining the second intron sequence (nucleotide sequence shown in SEQ ID NO.1) of rice LOG gene (gene="Os01g0588900"): Design PCR primers LOG-intron-F: (5'GAATTCAAGCTTGGATCCCTCGAGTCAAGGATTTCGGGATGACC) and LOG- intron-R (5'ACATGGGCCCAGATCTGTCGACTGGTCGCCATGTCA TTGG), using the genome of commercial rice variety Xiushui 134 as a template, a 0.7kb genome fragment was obtained by PCR amplification. PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 66°C for 15 seconds, 72°C for 1 minute, repeating 33 cycles; then 72°C for 10 minutes. Then, the fragment was cloned into pGEM-T easy Vector, verified by sequencing, and saved for subsequent transition vector construction. The vector was named pGEM-T-LOG-intron. The 5' end of the fragment obtained by above-mentioned PCR is provided with EcoRI, HindIII, BamHI, XhoI restriction sites successively, wherein EcoRI and HindIII are used for final vector construction, and BamHI and XhoI are respectively one of two groups of homozygous enzymes, used for The target fragment is connected; the 3' end is sequentially provided with SalI, BglII, and ApaI restriction sites, wherein SalI and BglII are the homologous enzymes of XhoI and BamHI, respectively, for connecting the target fragment, and ApaI is used to access the terminator.

The terminator is an artificial synthetic sequence Ter (SEQ ID NO. 2), the 5' end is provided with an ApaI restriction site, and the 3' end is provided with KpnI and SacI restriction sites.

Construction of transition vector containing intron and terminator: pGEM-T-LOG-intron was double digested with EcoRI and ApaI, and the LOG-intron fragment with a size of about 0.7kb was recovered; EcoRI and SacI were used for pGEM-T - LOG-intron was double digested to recover the pGEM-T vector with a size of about 3.0 kb; the artificially synthesized Ter that was linked into the pUC57 vector was digested with ApaI and SacI, and the terminator was recovered. Then, the recovered vector and fragment were connected in three sections, and the obtained vector was named pGEM-T-LOG-intron-Ter (Fig. 2), and the nucleotide sequence was shown in SEQ ID NO.3.

Example 2. Construction of RNAi vector for silencing rice OsTEL gene

The OsTEL (also named PLASTOCHRON2) gene in rice encodes an RNA-binding protein. Taiji et al. found that the rice with loss-of-function mutation of OsTEL gene had faster leaf growth rate, faster leaf maturation, and dwarf plants, indicating that it has the function of regulating leaf initiation and maturation (Kawakatsu, (2006) The Plant Cell 18: 612-625; Xiong et al., (2006) Cell Research 16:267-276). However, since the function of OsTEL gene in the above mutants is completely absent, the phenotype of OsTEL gene expression down-regulation and the corresponding relationship between different down-regulation degrees and phenotypes cannot be observed. Therefore, the construction of RNAi vector to silence OsTEL gene can help us to further study the function of OsTEL gene.

Obtainment of the target fragment (nucleotide sequence shown in SEQ ID NO. 4) in the OsTEL gene (gene="Os01g0907900"): Design PCR primers OsTEL-RNAi-F: (5'GACCTCGAGGGCTTCAGCATCGTCGTCTACC) and OsTEL-RNAi-R ( 5'CTTGGATCCGTCCGTGAGCAGCT TGCCGT), using the total cDNA of commercial rice variety Xiushui-134 as a template, a fragment with a size of 0.7kb was obtained by PCR amplification. PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 66°C for 15 seconds, 72°C for 1 minute, repeating 33 cycles; then 72°C for 10 minutes. Then, the fragment was cloned into pGEM-T easyVector, verified by sequencing, and saved for subsequent transition vector construction. The 5' end of the fragment obtained by the above PCR is provided with an XhoI restriction site, and the 3' end is provided with a BamHI restriction site.

Construction of the transition vector containing the target fragment targeting the OsTEL gene:

The first step: use BamHI and XhoI to double-enzymatically digest the pGEM-T easy Vector with the target fragment targeting the OsTEL gene, and recover the target fragment;

The second step: double-enzyme digestion was performed on the pGEM-T-LOG-intron-Ter vector constructed in Example 1 with BglII and SalI, and the vector fragments were recovered;

The third step: connect the recovered target fragment and the vector, and the constructed vector is named pGEM-T-LOG-intron-target fragment (inverted repeat)-Ter;

The fourth step: BamHI and XhoI double-enzymatically digest the pGEM-T-LOG-intron-target fragment (inverted repeat)-Ter vector, recover the vector fragment, and connect it with the fragment in the first step, the constructed The transition vector was named pGEM-T-target fragment-LOG-intron-target fragment (inverted repeat)-Ter.

Construction of RNAi final vector targeting OsTEL gene:

The binary vector pCambia1300-pZmUbi-G10 (Internationl Pat.No.PCT/CN2012/087069:SEQ ID NO.49) is modified from pCambia1300, and this vector contains a glyphosate-resistant gene (EPSPS) as a transformant marker gene. The pGEM-T-target fragment-LOG-intron-target fragment (inverted repeat)-Ter vector was double digested with BamHI and KpnI to obtain a target fragment containing "target fragment-LOG-intron-target fragment (inverted repeat)-Ter "fragment of the structure. The synthetic 35S promoter p35S (SEQ ID NO. 5, digested by HindIII and BamHI) was used to drive the transcription of the above fragment. The pCambia1300-pZmUbi-G10 vector digested by BamHI and KpnI, as well as the "target fragment-LOG-intron-target fragment (inverted repeat)-Ter" structural fragment and p35S recovered by enzyme digestion were connected in three sections to construct a final carrier. The vector T-DNA contains the following gene structure: "p35S-target fragment-LOG-intron-target fragment (inverted repeat)-Ter-promoter-glyphosate-tolerant gene". This vector was named: pCambia1300-G10-OsTEL-RNAi (Figure 3).

Example 3. Construction of RNAi vector for silencing maize Ms45 gene

The maize Ms45 gene is specifically expressed in top flowers, and maize plants with loss-of-function mutations in this gene are male sterile (Cigan et al., 2001). Only by constructing a high-efficiency RNAi vector and completely silencing the Ms45 gene can the standard for production and use be met.

Acquisition of the target fragment (nucleotide sequence shown in SEQ ID NO. 6) in the Ms45 gene (GenBank: AF360356.1): Design PCR primers MS45-RNAi-F: (5'GGTGCTCGAGCTCTAGATTAGTAAAAAGGGAGAGAGAGAG) and MS45-RNAi-R ( 5'TGGATCCTGCAGGTTCCTCTTCTCCATGCTGGTGGAC), using the genome of the commercial maize variety Zhengdan 958 as a template, a fragment of 0.4 kb in size was obtained by PCR amplification. PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 66°C for 15 seconds, 72°C for 30 seconds, repeating 33 cycles; then 72°C for 10 minutes. Then, the fragment was cloned into pGEM-Teasy Vector, verified by sequencing, and saved for subsequent transition vector construction. The 5' end of the fragment obtained by the above PCR is provided with an XhoI restriction site, and the 3' end is provided with a BamHI restriction site.

Construction of the transition vector containing the target fragment targeting the Ms45 gene:

The first step: use BamHI and XhoI to double-enzymatically digest the pGEM-T easy Vector with the target fragment targeting the Ms45 gene, and recover the target fragment;

The second step: double-enzyme digestion was performed on the pGEM-T-LOG-intron-Ter vector constructed in Example 1 with BglII and SalI, and the vector fragments were recovered;

The third step: connect the recovered target fragment and the vector, and the constructed vector is named pGEM-T-LOG-intron-target fragment (inverted repeat)-Ter;

The fourth step: BamHI and XhoI double-enzymatically digest the pGEM-T-LOG-intron-target fragment (inverted repeat)-Ter vector, recover the vector fragment, and connect it with the fragment in the first step, the constructed The transition vector was named pGEM-T-target fragment-LOG-intron-target fragment (inverted repeat)-Ter.

Construction of RNAi final vector targeting Ms45 gene:

The binary vector pCambia1300-pZmUbi-G10 (Internationl Pat.No.PCT/CN2012/087069:SEQ ID NO.49) is modified from pCambia1300, and this vector contains a glyphosate-resistant gene (EPSPS) as a transformant marker gene. The pGEM-T-target fragment-LOG-intron-target fragment (inverted repeat)-Ter vector was digested to obtain a fragment containing the structure of "target fragment-LOG-intron-target fragment (inverted repeat)-Ter". The synthetic 35S promoter p35S (SEQ ID NO. 5, digested by HindIII and BamHI) was used to drive the transcription of the above fragment. The pCambia1300-pZmUbi-G10 vector digested by BamHI and KpnI, as well as the "target fragment-LOG-intron-target fragment (inverted repeat)-Ter" structural fragment and p35S recovered by enzyme digestion were connected in three sections to construct a final carrier. The vector T-DNA contains the following gene structure: "p35S-target fragment-LOG-intron-target fragment (inverted repeat)-Ter-promoter-glyphosate-tolerant gene". This vector was named: pCambia1300-G10-Ms45-RNAi (Figure 3).

Example 4. Transformation of rice

The method of obtaining transgenic rice is to use the existing technology (Lu Xiongbin, Gong Zuxun (1998) Life Science 10: 125-131; Liu Fan et al. (2003) Molecular Plant Breeding 1: 108-115). The mature and plump "Xiushui 134" seeds were selected to be shelled, and callus was induced to be used as the transformation material. Take the vectors constructed in Example 2 and carry out Agrobacterium streaking respectively. Pick a single colony to inoculate and prepare for transformation with Agrobacterium. The callus to be transformed is put into the recombinant Agrobacterium liquid with an OD of about 0.6 (preparation of the recombinant Agrobacterium liquid: the recombinant Agrobacterium is inoculated into the medium, and cultivated at 28° C. to an OD of about 0.6; the medium consists of : 3g/LK ₂ HPO ₄ , 1g/L NaH ₂ PO ₄ , 1g/L NH ₄ Cl, 0.3g/L MgSO ₄ ·7H ₂ O, 0.15g/L KCl, 0.01g/LCaCl ₂ , 0.0025g/L FeSO ₄ ·7H ₂ O, 5g/L sucrose, 20mg/L acetosyringone, the solvent is water, pH=5.8), let the recombinant Agrobacterium bind to the surface of the callus, and then transfer the callus to the co-cultivation medium (MS+2mg/L 2,4-D+30g/L glucose+30g/L sucrose+3g/L agar (sigma 7921)+20mg/L acetosyringone), co-culture at 28°C for 2-3 days. The transformed calli were washed with sterile water and transferred to screening medium (MS+2mg/L 2,4-D+30g/L sucrose+3g/L agar (sigma 7921)+20mg/L acetosyringone+2mM Glyphosate (Sigma)), screen culture at 28°C for two months (one intermediate passage). Transfer the callus with good growth vigor after screening to pre-differentiation medium (MS+0.1g/L inositol+5mg/L ABA+1mg/L NAA+5mg/L 6-BA+20g/L sorbitol+30g/L) L sucrose + 2.5 g/L gelrite) were cultured at 28°C for about 20 days, and then the pre-differentiated callus was transferred to the differentiation medium, and differentiated and germinated by light for 14 hours a day. After 2-3 weeks, the resistant regenerated plants were transferred to rooting medium (1/2MS+0.2mg/L NAA+20g/L sucrose+2.5g/L gelrite) for strong seedlings and rooting, and finally the regenerated plants were washed off the agar Transgenic lines with high yield, large seeds or high biomass that can improve rice yield are selected for transplanting in the greenhouse, and new varieties are cultivated. A transgenic rice plant containing the above transformation vector is obtained.

Example 5. Identification of transgenic rice

Rice is an inbred line, and we used the obtained target gene transgenic rice line and the homozygote of the empty vector control line to analyze and compare the phenotype.

The number of leaves and plant height of the transgenic rice line obtained in Example 4 and the non-transgenic recipient line were compared and analyzed.

We obtained 60 transgenic lines (named OsTEL-i) transfected with pCambia1300-G10-OsTEL-RNAi vector, 45 lines were faster than the non-transgenic control in terms of initial leaf development rate, leaf maturity, and plant height became shorter.

The changes in leaf number and plant height of two typical lines are shown in the table below.

Table 1: Comparison of two representative lines with a non-transgenic control line

OsTEL-i-20 OsTEL-i-55 Number of leaves twenty one% 32% Plant height -32% -49%

*The parameters in the table are all tested by F-Test (P≤0.05), and the difference is more than 5% compared with the control. OsTEL-i is a T-DNA transformed plant of the vector pCambia1300-G10-OsTEL-RNAi, wherein the numbers (20, 55) behind OsTEL-i are random numbers for different lines to distinguish different transformation events.

Example 6. Transformation of maize

The transformation technology of maize is relatively mature. References such as: Vladimir Sidorov & David Duncan (in M. Paul Scott (ed.), Methods in Molecular Biology: Transgenic Maize, vol: 526; Yuji Ishida, Yukoh Hiei & Toshihiko Komari (2007) Agrobacterium-mediated transformation of maize. Nature Protoc ols 2:1614- 1622. The basic method is as follows:

Hi-II corn ears 8-10 days after pollination were taken and all immature embryos (1.0-1.5 mm in size) were collected. The recombinant Agrobacterium and immature embryos obtained by transforming Agrobacterium with the T-DNA vector constructed in Example 3 were cultured on a co-cultivation medium (MS+2mg/L 2,4-D+30g/L sucrose+3g/L Agar (sigma 7921) + 40 mg/L acetosyringone) was co-cultured for 2-3 days (22°C). Transfer immature embryos to callus induction medium (MS+2mg/L2,4-D+30g/L sucrose+2.5g/L gelrite+5mg/L AgNO ₃ +200mg/L acetosyringone), dark at 28°C Culture for 10-14 days. All calli were transferred to selection medium (same as callus induction medium) with 2 mM glyphosate and cultivated in the dark at 28°C for 2-3 weeks. All tissues were transferred to fresh selection medium containing 2 mM glyphosate and incubated in the dark at 28°C for 2-3 weeks. Then, transfer all the viable embryogenic tissues after selection to regeneration medium (MS+30g/L sucrose+0.5mg/L kinetin+2.5g/L gelrite+200mg/L acetosyringone), and culture at 28°C in the dark for 10- 14 days, one strain per dish. Embryogenic tissue was transferred to fresh regeneration medium and incubated in the light at 26°C for 10-14 days. All fully developed plants were transferred to rooting medium (1/2MS+20g/L sucrose+2.5g/L gelrite+200mg/L acetosyringone) and cultivated in the light at 26°C until the roots were fully developed. Transgenic maize plants containing the above transformation vector are obtained.

Example 7. Identification of transgenic corn

We pollinated and harvested the transgenic maize obtained in Example 6 using the method of artificial pollination, and then planted the harvested T1 generation seeds for observation and statistical analysis.

Of the 58 transgenic lines we obtained (named Ms45-i) transfected with the pCambia1300-G10-Ms45-RNAi vector, 28 were completely male sterile, 20 were partially male sterile, and 10 had fertility comparable to the non-transgenic control .

Claims (5)

1. An RNAi vector constructed based on isocaudarner, which is characterized in that the construction method of the vector is as follows: introducing a target fragment of the targeted gene into a transition vector to construct an RNAi vector capable of generating hairpin-structure RNA;

an intron and a terminator are preset in the transition vector, isocaudarner enzyme cutting sites A1 and B1 are sequentially arranged at the 5 'end of the intron, isocaudarner enzyme cutting sites B2 and A2 are sequentially arranged at the 3' end of the intron, wherein A1 and A2 are a group of isocaudarner enzyme cutting sites, and B1 and B2 are the other group of isocaudarner enzyme cutting sites; the 5 'end and the 3' end of the target fragment are respectively provided with an A1 enzyme cutting site, a B1 enzyme cutting site, an A2 enzyme cutting site and a B2 enzyme cutting site; respectively connecting a target fragment to the 5 'end and the 3' end of an intron through two times of enzyme digestion, connection and transformation reactions to form a DNA fragment with a structure of 'target fragment-intron-inverted repetitive target fragment-terminator', and connecting the DNA fragment to a terminal vector with a preset promoter through one time of enzyme digestion, connection and transformation reactions, or connecting the DNA fragment to the terminal vector through three sections of connection with the designed promoter to obtain an RNAi vector; the intron nucleotide sequence is shown in SEQ ID NO. 1; the isocaudarner is one of the following: (1) BglII and BamHI, (2) NheI and XbaI, (3) SalI and XhoI; the final vector is a binary vector pCambia 1300-pZmUbi-G10; the target gene is rice OsTEL gene or maize Ms45 gene.

2. The RNAi vector constructed based on a isocaudarner of claim 1 wherein the terminator nucleotide sequence is represented by SEQ ID No. 2.

3. The RNAi vector of claim 1, constructed based on the isocaudarner, wherein the construction method comprises:

(1) taking pGEM-T easy Vector as a framework, connecting an intron and a terminator into the Vector, and simultaneously sequentially arranging enzyme cutting sites A1, B1, B2 and A2 at two ends of the intron to form a transition Vector 1;

(2) carrying out enzyme digestion recovery on target genes of which two ends obtained by PCR amplification are respectively provided with enzyme digestion sites A1 and B1 or A2 and B2 to obtain target fragments;

(3) then, carrying out enzyme digestion and recovery on the transition vector 1 by using two isocaudarner enzymes arranged on a target gene to obtain a vector;

(4) connecting and transforming the target fragments recovered in the step (2) and the step (3) with a vector to obtain a transition vector 2 containing a section of target gene;

(5) carrying out enzyme digestion recovery on the transition vector 2 by using corresponding isocaudarner provided with enzyme digestion sites of the same target gene, and carrying out connection transformation on the transition vector and the target fragment recovered in the step (2) to obtain a transition vector 3 with a structure of target fragment-intron-reverse repeated target fragment;

(6) and finally, carrying out enzyme digestion and recovery on the structure of the target fragment-intron-inverted repetitive target fragment and the terminator in the transition vector 3, and then connecting the structure and the terminator into a corresponding final vector to obtain the RNAi vector.

4. Use of the isocaudarner-based RNAi vector of claim 1 for gene transformation.

5. Use of the RNAi vector constructed based on the isocaudarner of claim 1 in the preparation of transgenic plants.

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