Rice black-streaked dwarf virus rnai polyvalent target gene sequence and application

Abstract

The invention relates to a preferred rice black-streaked dwarf virus RNAi multivalent target gene sequence which sequentially comprises 206 bp segment in a rice black-streaked dwarf virus S8 code region, 205 pb in an S10 code region, 203 pb in an S4 code region, wherein a BamH1 restriction enzyme cutting site and three CCG protective bases are added at the 5'end of the S8; an XhoI restriction enzyme cutting site and three ACA protective bases are added at the 3'end of S4. According to the rice black-streaked dwarf virus RNAi multivalent target gene sequence and application, based on a special design and optimization and specific to common 614 bp of RANi target sequence of three genes, namely RBSDV S8, S10, and S4, the specific rice black-streaked dwarf virus RNAi multivalent target gene sequence is acquired, cloned and structured into an RNAi carrier pBDL03 to obtain an RNA silent carrier pBDL03-S8+S10+S4; then RNA silent carrier pBDL03-S8+S10+S4 is transferred to a rice variety Taigeng 394 through an agrobacterium transformation method. T1 generation positive plants have favorable resistance on RBSDV.

Description

technical field

[0001] The invention relates to the field of biotechnology, in particular to a method and application for constructing an anti-rice black-streaked dwarf virus vector using RNAi-mediated technology. Background technique

[0002] Rice black-streaked dwarf virus (RBSDV) belongs to the Fijivirus genus of the Reoviridae family, and is mainly transmitted by persistent non-ovipositous means by the gray planthopper (Laodelphax striatellus fallen) (Milne and Lovisolo, 1977; Azuhata et al., 1993). The virus mainly causes rice black-streaked dwarf and maize rough dwarf, and can infect rice, barley, wheat, corn and other gramineous plants. RBSDV was first discovered in Japan in the 1940s, and then became popular in North China and East my country in the 1960s and 1990s, causing serious economic losses (Xu Qiufang et al., 2012). In recent years, the harm of RBSDV has become more and more serious year by year, causing serious economic losses. The incidence area of ​​the...

Examples

Embodiment 1 3

[0017] Nucleotide sequence and cloning of embodiment 1 trivalent RNAi gene fragment

[0018] According to the RBSDV sequence submitted by NCBI, multiple sequence alignment and restriction enzyme analysis were performed on DNAMAN6.0.3.99, and the relatively conserved S8, S10, and S4 coding region genes that did not contain BamHI and XhoI restriction sites were selected For fragments, the online design software of Wuhan Jingsai Bioengineering Technology Co., Ltd. was used to predict the siRNA target site, and the off-target effect was predicted in the rice genome database (http: / / signal.salk.edu / cgi-bin / RiceGE). Optimized to obtain 614bp RNAi multivalent target gene sequence (such as figure 1 shown; as shown in SEQ ID NO.1), which in turn includes the 206bp fragment of the S8 coding region (corresponding to S8 gene 30~131bp, 1015~1118bp), the 205bp of the S10 coding region (corresponding to the S10 gene 433~637bp), S4 203bp of the coding region (corresponding to 2218~2420bp of ...

Embodiment 2 3

[0019] Example 2 Construction of Trivalent RNAi Plant Expression Vector

[0020] The concentration and purity of the entry vector tENTRA-S2+S6+S10 and the destination vector pBDL03 were detected by ultraviolet spectrophotometer and agarose gel electrophoresis, and then the entry vector and the destination vector were subjected to LR reaction at a ratio of 1:1. According to Gat LR Clonase TM II Enzyme Mix (Invitrogen) kit instructions Construct 10ul reaction system, overnight at 25°C, add 1ul Proteinase K solution, treat at 37°C for 10min to terminate the reaction. Take 1ul of the above reaction solution to transform trans5α competent cells, after centrifugation, spread it evenly on a solid LB plate containing 50mg / L kanamycin, pick a single clone for plasmid extraction and double enzyme digestion verification ( figure 2 ), and designed primers GUS3 (5'-CAGTCCATTAATGCGTGGTCGT-3') and GUS4 (5'-TGTATCACCGCGTCTTTGATCG-3') according to the Gus-linker sequence on the target vec...

Embodiment 3

[0021] Embodiment 3 Agrobacterium transformation and the acquisition of transgenic rice

[0022] The RNAi plant expression vector pBDL03-S8+S10+S4 was transformed into the rice variety Taijing 394 by the Agrobacterium-mediated method, and the specific experimental steps were as follows:

[0023] (1) Induction of callus

[0024] a: select fine rice mature seeds and shell them into brown rice. Transfer an appropriate amount of brown rice to a 50ml centrifuge tube (the following steps require aseptic operation), add 75% ethanol solution to infiltrate and wash the brown rice for 2 minutes, pour off the ethanol solution, and rinse the seeds twice with sterile water.

[0025] b: Wash the seeds once with 2% sodium hypochlorite with available chlorine, and shake the centrifuge tube intermittently for 30 minutes. Pour off the sodium hypochlorite solution, rinse the seeds with sterile water 4 times, five minutes each time for the first three times, soak for 30 minutes for the last tim...

technical field

[0001] The invention relates to the field of biotechnology, in particular to a method and application for constructing an anti-rice black-streaked dwarf virus vector using RNAi-mediated technology. Background technique

[0002] Rice black-streaked dwarf virus (RBSDV) belongs to the Fijivirus genus of the Reoviridae family, and is mainly transmitted by persistent non-ovipositous means by the gray planthopper (Laodelphax striatellus fallen) (Milne and Lovisolo, 1977; Azuhata et al., 1993). The virus mainly causes rice black-streaked dwarf and maize rough dwarf, and can infect rice, barley, wheat, corn and other gramineous plants. RBSDV was first discovered in Japan in the 1940s, and then became popular in North China and East my country in the 1960s and 1990s, causing serious economic losses (Xu Qiufang et al., 2012). In recent years, the harm of RBSDV has become more and more serious year by year, causing serious economic losses. The incidence area of ​​the...

Examples

Embodiment 1 3

[0017] Nucleotide sequence and cloning of embodiment 1 trivalent RNAi gene fragment

[0018] According to the RBSDV sequence submitted by NCBI, multiple sequence alignment and restriction enzyme analysis were performed on DNAMAN6.0.3.99, and the relatively conserved S8, S10, and S4 coding region genes that did not contain BamHI and XhoI restriction sites were selected For fragments, the online design software of Wuhan Jingsai Bioengineering Technology Co., Ltd. was used to predict the siRNA target site, and the off-target effect was predicted in the rice genome database (http: / / signal.salk.edu / cgi-bin / RiceGE). Optimized to obtain 614bp RNAi multivalent target gene sequence (such as figure 1 shown; as shown in SEQ ID NO.1), which in turn includes the 206bp fragment of the S8 coding region (corresponding to S8 gene 30~131bp, 1015~1118bp), the 205bp of the S10 coding region (corresponding to the S10 gene 433~637bp), S4 203bp of the coding region (corresponding to 2218~2420bp of ...

Embodiment 2 3

[0019] Example 2 Construction of Trivalent RNAi Plant Expression Vector

[0020] The concentration and purity of the entry vector tENTRA-S2+S6+S10 and the destination vector pBDL03 were detected by ultraviolet spectrophotometer and agarose gel electrophoresis, and then the entry vector and the destination vector were subjected to LR reaction at a ratio of 1:1. According to Gat LR Clonase TM II Enzyme Mix (Invitrogen) kit instructions Construct 10ul reaction system, overnight at 25°C, add 1ul Proteinase K solution, treat at 37°C for 10min to terminate the reaction. Take 1ul of the above reaction solution to transform trans5α competent cells, after centrifugation, spread it evenly on a solid LB plate containing 50mg / L kanamycin, pick a single clone for plasmid extraction and double enzyme digestion verification ( figure 2 ), and designed primers GUS3 (5'-CAGTCCATTAATGCGTGGTCGT-3') and GUS4 (5'-TGTATCACCGCGTCTTTGATCG-3') according to the Gus-linker sequence on the target vec...

Embodiment 3

[0021] Embodiment 3 Agrobacterium transformation and the acquisition of transgenic rice

[0022] The RNAi plant expression vector pBDL03-S8+S10+S4 was transformed into the rice variety Taijing 394 by the Agrobacterium-mediated method, and the specific experimental steps were as follows:

[0023] (1) Induction of callus

[0024] a: select fine rice mature seeds and shell them into brown rice. Transfer an appropriate amount of brown rice to a 50ml centrifuge tube (the following steps require aseptic operation), add 75% ethanol solution to infiltrate and wash the brown rice for 2 minutes, pour off the ethanol solution, and rinse the seeds twice with sterile water.

[0025] b: Wash the seeds once with 2% sodium hypochlorite with available chlorine, and shake the centrifuge tube intermittently for 30 minutes. Pour off the sodium hypochlorite solution, rinse the seeds with sterile water 4 times, five minutes each time for the first three times, soak for 30 minutes for the last tim...

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