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Functional Analysis of Jatropha Genes

一种麻风树属、基因的技术，应用在基因工程、植物基因改良、生物化学设备和方法等方向，能够解决不适用高通量分析、费力费时等问题

Inactive Publication Date: 2011-12-07
TEMASEK LIFE SCIENCES LABORATORY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene function can be more accurately determined by overexpression or RNAi-mediated silencing of candidate genes in transgenic plants; however, such methods are laborious, time-consuming and not suitable for genome-scale high-throughput analysis

Method used

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  • Functional Analysis of Jatropha Genes
  • Functional Analysis of Jatropha Genes
  • Functional Analysis of Jatropha Genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1 Experimental method

[0090] Jatropha seedlings: Jatropha seeds purchased from India were germinated in a greenhouse. Jatropha seedlings 10-14 days after germination were used for the VIGS method. At this stage, the seedlings have 2-3 true leaves.

[0091] Original TRV vectors: pTRV1 (GenBank No.AF406990) and pTRV2 (GenBank No.AF406991) were kindly provided by Dr. Dinesh Kumar (Yale University).

[0092] Synthetic TRV RNA1 expression vector: The full-length (7756bp) sequence of the synthetic TRV1 vector includes: SphI site, T-DNA right border sequence (152bp), repeated cauliflower mosaic virus (CaMV) 35S enhancer region (752bp) (Shi et al. al., 1997), TRV Ppk20 strain RNA1 (6791bp), subterranean clover mottle virus satellite RNA ribozyme sequence (46bp) and SmaI site sequence. The endogenous SalI site divides the full-length sequence into two parts. These two parts were synthesized separately and cloned into pGH vector to obtain two vectors pGH-YeJ-V1-1...

Embodiment 2

[0105] Example 2 Development of a VIGS system using CH42 as a marker gene in Jatropha curcas

[0106] This example describes the construction of a tobacco rattle virus (TRV)-based vector and its use in gene silencing of Jatropha curcas. Virus-induced gene silencing (VIGS) is initiated when a recombinant virus carrying a sequence derived from a host gene infects a plant. Transcripts of endogenous genes with sequences homologous to the insert in VIGS vectors are degraded by post-transcriptional gene silencing mechanisms (PTGS) (Baulcombe, 2004).

[0107] Initially, a TRV vector kindly provided by Dr. Dinesh Kumar (Yale University) was used in this study. TRV is a bisense RNA virus. TRV RNA1 encodes 134 kDa and 194 kDa replicase proteins from genomic RNA, a 29-kDa mobile protein, and a 16-kDa cysteine-rich protein from subgenomic RNA. TRV RNA2 encodes a coat protein from genomic RNA and two nonstructural proteins from subgenomic RNA. In the absence of RNA2, TRV RNA1 can repli...

Embodiment 3

[0113] Example 3 Optimization of a VIGS system by vacuum infiltration

[0114] Following the CH42 gene, we next chose to silence another marker gene, phytoene desaturase (PDS), which encodes a key enzyme involved in carotenoid biosynthesis. Silencing of the PDS gene inhibits carotenoid biosynthesis, which causes chlorophyll photooxidation and destruction under high light intensities and leads to photobleached leaves.

[0115] To amplify PDS homologs from Jatropha, PCR primers were designed to target conserved sequences of PDS from different species of Euphorbiaceae. First, we obtained the 302-bp PDS cDNA of Jatropha curcas by PCR and inserted this fragment into pTRV2 to obtain pTRV2-PDSS. Using this short PDS sequence as a seed, the longer EST sequences of different Euphorbiaceae PDS genes were searched in GenBank by BLASTN. We further designed PCR primers to clone the 786-bp fragment of Jatropha PDS cDNA to generate pTRV2-PDSL.

[0116] A mixture of Agrobacterium cultures ...

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Abstract

The present invention relates to the field of functional analysis of Jatropha genes on a genomic scale. More specifically, the present invention relates to a method for high-throughtput functional analysis of Jatropha curcas genes on a genomic scale using virus-induced gene silencing. The method involves use of the tobacco rattle virus (TRV).

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 61 / 143,484, filed January 9, 2009, which is incorporated herein by reference. Background of the invention [0003] The invention relates to the field of genome-scale Jatropha gene function analysis. More specifically, the present invention relates to a method for genome-scale high-throughput functional analysis of Jatropha curcas genes by using virus-induced gene silencing. [0004] The publications and other materials used herein to illustrate the background of the invention or to provide additional details pertaining to its practice are incorporated herein by reference and are grouped separately in the bibliography for convenience. [0005] The world faces dwindling fossil fuel supplies and deteriorating greenhouse effects. There is an urgent need to increase the generation and consumption of renewable energy. Biofuels have been recognized as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/83A01H1/06
CPCC12N15/8218
Inventor 叶健蔡南海曲景
Owner TEMASEK LIFE SCIENCES LABORATORY
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