RNAi (RNA interference) vector against barley yellow dwarf virus and wheat dwarf virus as well as construction method and application thereof in wheat genetic transformation
A technology of barley yellow dwarf virus and wheat dwarf virus, which is applied in the field of molecular biology and can solve problems that have not been reported
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Embodiment 1
[0078] Example 1. Construction of RNA interference vectors with double resistance to barley yellow dwarf virus and wheat dwarf virus
[0079] Step 1, according to the genome sequence of BYDV-GAV, the present invention first designs full-length CP gene primers, as follows:
[0080] SEQ ID NO1: (BYDV-GAV-CP) 5'-ATGAATTCAGTAGGCCGTAGA-3'
[0081]SEQ ID NO2: (BYDV-GAV-CP)5'-CTATTTGGGAGTCATGTTGGC-3'
[0082] Using the total RNA of BYDV-GAV as a template, the full-length CP gene fragment (600bp) was amplified by RT-PCR ( figure 1 ), the target fragment was recovered and then connected to the pGEM T-easy vector, transformed into E. coli DH5α, cloned and sequenced for identification, and a positive clone of the BYDV-GAV-CP gene was obtained.
[0083] Step 2, according to the genome sequence of WDV, the present invention designs the primer of full-length CP gene, as follows:
[0084] SEQ ID NO3: (WDV-CP) 5'-ATGGTGACCAACAAGGACTCCC-3'
[0085] SEQ ID NO4: (WDV-CP) 5'-TTACTGAATGCCGATG...
Embodiment 2
[0107] The acquisition of embodiment 2 transgenic plants ( Figure 8 )
[0108] Step 1. The interference vector and its control vector were respectively transformed into competent cells DH 5α, plated, and cultured at 37°C overnight. Pick a single colony, shake and cultivate a small amount of liquid LB (3-5ml); after a medium amount of culture (20-30ml); inoculate 200ml of liquid LW medium at a ratio of 1:50, and shake and cultivate at 37 ° C for 2-3 hours; Chloramphenicol with a final concentration of 140 μg / ml was added, and the culture was continued for 16-18 hours; the cells were collected by centrifugation at 5000 rpm for 10 minutes; the plasmid was purified by a large amount of plasmid extraction kit (TIANGEN). See the manufacturer's instructions for the method. The concentration of the obtained plasmid reaches 2-3 mg / ml, contains more than 90% supercoiled DNA, and can be used for gene gun transformation.
[0109] Step 2. Collect young wheat ears (variety: Yangmai 158,...
Embodiment 3
[0112] Example 3 Molecular detection of transgenic wheat plants (Figure 9)
[0113] In the present invention, SEQ ID NO1 / SEQ ID NO2, SEQ ID NO3 / SEQ ID NO4, SEQ ID NO8 / SEQ ID NO8, SEQ ID NO10 / SEQ ID NO11 are used as primers, and Yangmai 158, Yangmai 12 and Yangmai 158 are used as primers. 518 strains of T as receptors 0 PCR detection was carried out on the transgenic regenerated plants, and the number of T0 transgenic wheat positive plants of pSA, pS, pA and pMCG161 were 28, 8, 6 and 4, respectively. The obtained T1 and T2 and transgenic regenerated plants were tracked and detected, and the transgenic progeny plants showed a 3:1 segregation.
[0114] The test results are shown in Figure 9: Figure 9A It is the PCR detection of the full length (783bp) of WDV-CP in the transgenic progeny; Figure 9B It is the PCR detection of intron (513bp) in the transgenic progeny; Figure 9C It is the PCR detection of the full length (600bp) of BYDV-CP in the transgenic offspring; Figure...
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