TYLCV and ToCV bivalent RNAi expression vector established through Gateway technology and application of expression vector

A technology of expression vector, prnai-tocv-ty, applied in the field of genetic engineering

A technology of expression vector, prnai-tocv-ty, applied in the field of genetic engineering

CN110295192AActive Publication Date: 2019-10-01天津市农业科学院
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  • TYLCV and ToCV bivalent RNAi expression vector established through Gateway technology and application of expression vector
  • TYLCV and ToCV bivalent RNAi expression vector established through Gateway technology and application of expression vector
  • TYLCV and ToCV bivalent RNAi expression vector established through Gateway technology and application of expression vector

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Embodiment 1

[0051] 1 Materials and methods

[0052] 1.1 Materials

[0053] In the tomato cultivation field of the Sixth Fu solar greenhouse in Xiqing District, Tianjin, tomato leaves with typical characteristics of tomato yellow leaf curl virus and tomato chlorosis virus were collected for subsequent tests of nucleic acid extraction. The tomato susceptible inbred line J501 was preserved by our laboratory.

[0054] pDONR TM 221 Vector, BP Clonase II enzyme Mix, LR Clonase II enzyme Mix, and reverse transcriptase were purchased from Invitrogen, USA. rTaq DNA polymerase, dNTPs, DNA recovery and purification kit, and RNA extraction kit were purchased from Dalian Bao Biological Engineering Co., Ltd., and the plasmid mini-extraction kit and Agrobacterium GV3101 were purchased from Tiangen Biochemical Technology Co., Ltd. PCR primers were synthesized by Dalian Bao Biological Engineering Co., Ltd. pK7GWIWG2(I), 2×SG PCRMaster Mix are products of Beijing Xinnuo Jinda Biotechnology Co., Ltd. T...

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Abstract

The invention discloses a TYLCV and ToCV bivalent RNAi expression vector established through a Gateway technology and application of the expression vector. The bivalent RNAi expression vector is namedas pRNAi-TOCV-TY. A nucleotide sequence of the RNAi expression vector is shown in SEQ ID NO.5. According to the expression vector, an RNAi technology is used for a lot of research work, through the Gateway technology, the TYLCV and ToCV coat protein gene bivalent RNAi expression vector is established, and through experiment methods such as genetic transformation and resistance identification, a transgenic lycopersicon esculentum Mill. plant resisting the two viruses is obtained. The invention further discloses application of the pRNAi-TOCV-TY expression vector in effective prevention and control over lycopersicon esculentum Mill. with composite infection of the ToCV and TYLCV, particularly application of the pRNAi-TOCV-TY expression vector in effective obstruction for lycopersicon esculentum Mill. virus replication in plant bodies.

Description

technical field [0001] The present invention provides a fast and simple construction of anti-TYLCV and ToCV virus bivalent RNAi expression vector by using Gateway technology, and is used to inhibit the infection of these two viruses, thereby cultivating a new plant material with double antibodies. It belongs to the technical field of genetic engineering. Background technique [0002] tomato( Lycopersicon esculentum Mill.) is one of the main vegetable crops under facility cultivation in my country, and plays an important role in the annual supply of vegetables. However, with the increasing tomato planting area and the diversification of introduction channels, tomato virus diseases are also becoming more and more serious, which has become a bottleneck restricting the development of tomato. [0003] Currently, tomato chlorotic virus ( Tomato chlorosis virus , ToCV) and tomato yellow leaf curl virus ( Tomato yellow leaf curl virus, TYLCV ) is the most serious damage to tom...

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Application Information

Patent Timeline
01 Oct 2019
Publication
CN110295192A
IPC
C12N15/82; A01H5/00; A01H6/82
CPC
C07K14/415; C12N15/8218; C12N15/8283
Inventors
金凤媚; 宋建