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Eggplant fruit gene silencing system and construction method thereof

A technique for eggplant fruit and gene silencing, applied in the fields of molecular biology and plant genetic engineering

Active Publication Date: 2019-05-28
INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] According to previous reports, virus-induced gene silencing in eggplant is basically based on cotyledons (Liu HP, FDQ, Zhu BZ, et al. Virus-induced gene silencing in eggplant (Solanum melongena L.). Journal of Integrative Plant Biology,2012,54(6):422-429; Zhao Zhen, Liu Fuzhong, Zhang Ying, etc. Construction and expression analysis of eggplant Sm MsrA gene VIGS expression vector. Acta Horticultural Science, 2015, 42(8):1495-1504) as Receptors, direct use of eggplant fruit as VIGS receptor tissue has not yet been reported

Method used

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  • Eggplant fruit gene silencing system and construction method thereof
  • Eggplant fruit gene silencing system and construction method thereof
  • Eggplant fruit gene silencing system and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0055] 1. Test materials, VIGS vectors and genes

[0056] 1. Plant materials: The hybrid combination 3316F1 selected by the Guangdong Academy of Agricultural Sciences Vegetable Research, its hypocotyls, leaf veins, flower color and fruit all contain anthocyanins, and the skin color is purple red ( figure 1 ). Plug seedlings were raised on July 10, 2018, planted in the open field on August 10, and VIGS infection experiments were carried out on young fruits during the eggplant period on October 10.

[0057] 2. Vector: Silencing vector pTRV-RNA2 (pYL156) and helper vector pTRV-RNA1 (pYL192) were gifted by Professor Dinesh-Kumar from the DAVIS campus of the University of California and kept in our laboratory.

[0058] 3. Silence the target gene: The sequence is derived from the eggplant transcriptome data (unpublished), from which a key structural gene encoding the anthocyanin metabolism pathway of eggplant, the anthocyanin synthase gene SmANS Unigene0020595 (such as SEQ ID NO.1) was sel...

Embodiment 2

[0084] Application of VIGS System in Eggplant Fruit

[0085] 1. Silencing effect of SmANS gene in eggplant peel

[0086] (1) The effect of structural gene SmANS silencing on phenotypic traits of eggplant peel color

[0087] From image 3 B. It can be seen that the eggplant fruit infected by the negative control pTRV2 (equal volume of TRV1 and TRV2) Agrobacterium infected is compared with the uninfected fruit of the control ( image 3 A) The fruit type has changed slightly, but the color of the peel has not changed. SmANS, a key structural gene in the anthocyanin metabolism pathway, constructs VIGS vectors pTRV2-SmANS-1 and pTRV2-SmANS-2 in two different target segments. After Agrobacterium transformation, the synthesis of anthocyanin in the peel around the infected area is affected. The color of the peel changed from purple to white ( image 3 C, image 3 D), indicating that the SmANS gene in the eggplant peel was effectively silenced.

[0088] (2) qPCR detection of the effects of di...

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Abstract

The invention discloses an eggplant fruit gene silencing system and a construction method thereof and in particular discloses application of CDS sequences of eggplant anthocyanin anabolism related genes including SmANS and SmMYB1 genes to construction of an eggplant fruit VIGS system; the CDS sequences are shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention establishes an efficient and rapid eggplant fruit virus mediated gene silencing system which can be used for verifying and analyzing fruit important property regulation and control gene functions including eggplant fruit peel pigment metabolism, peel color and luster, parthenocarpy and the like. Influences, caused by environment temperature, sizes of fruits for injection, sizes of target gene insert segments and injection amount, on the eggplant fruit VIGS system are defined. The gene silencing system disclosed by the invention is simple, rapid and efficient and can be applied to eggplant fruit development and functional verification of other tissue genes containing anthocyanin.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and particularly relates to the technical field of plant genetic engineering, in particular to an eggplant fruit gene silencing system and a construction method thereof. Background technique [0002] Virus Induced Gene Silence (VIGS) is used to reveal the RNA-mediated antiviral defense mechanism. It is a post-transcriptional gene silencing (PTGS) phenomenon, which is inherent in plants. The ubiquitous genetic immune mechanism. When the unmodified virus infects plants, its mechanism is to specifically target the viral genome; but when the host gene segment is inserted into the virus vector, its target of attack increases the mRNA corresponding to the plant host gene segment. VIGS, as a reverse genetics technology, has been widely used in the study of gene functions such as plant development, biological adversity, abiotic adversity, cell metabolism, and signal transduction in the past 20 years. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/29C12N15/82C12N15/11
Inventor 李植良孙保娟孙光闻李慧敏吴俊李涛衡周宫超黎振兴李颖王恒明徐小万
Owner INST OF VEGETABLES GUANGDONG PROV ACAD OF AGRI SCI
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