Improve The Oil Content Of Rapeseed (brassica Napus. L) By Rna Interference

Rape is one of the world's major oil crops, and is an important source of edible oil in China. China's consumption of edible oil comes from rapeseed oil more than 40%. China is one of big country in rapeseed cultivation, and area and output ranks first in the world. There are about 1 million hectares in rapeseed cultivation in China each year, about 14 million tons of rapeseed production, oil content 38%, providing about 5 million tons edible oil. China's oil consumption every year the gap in more than 11 million tons, increased the oil content of rapeseed variety, is the imperative to solve the shortage of edible oil in China. China's rapeseed oil was lower than Canada, Australia, and EU, rapeseed imports were mainly from Canada and Australia.In recent years, biotechnology rapidly developed, particularly transgenic technology, gene expression interference technology (RNAi), and they provide advanced technical support for crop resources innovation, breeding and gene expression studies. They reduce the breeding cycle, reduce costs, and improve efficiency. Japanese scientists first discovered a negative correlation fatty between protein in soybean and rapeseed. A Chinese scholar, Chen Jin-Qing, proposed the substrate competition hypothesis of a rapeseed protein and fat accumulation by this principle. According to this hypothesis, pyruvate(PEP) is a common substrate of protein and fatty acid synthesis, and protein and oil complete the accumulation through the competition to pyruvate. During seed development in rapeseed, sugar produce by photosynthesis, and sugar become the pyruvate by glycolysis (PEP). The pyruvate become oxaloacetate through the catalysis of phosphoenol pyruvate carboxylase (PEPC), and then the oxaloacetate enter the protein metabolism pathway. The pyruvate generate acetyl-CoA through the catalysis of pyruvate dehydrogenase, and acetyl-CoA is the precursor of fatty acid synthesis and ultimately into the fatty acid biosynthesis metabolism pathway. Thus, inhibition of protein synthesis can be increased the accumulation of oil to some extent. Pyruvate by Phosphoenolpyruvate carboxylase (PEPC) catalysts enter protein pathway, and therefore inhibition the PEPC gene expression, can suppress the protein accumulation, ultimately improve the oil content.In this study, by RNA interference (RNAi) technology, the sequence of PEPC activity site was cloned from rapeseed (Brassica napus.L) to construct ihpRNA genetic expression vector. The expression vector was introduced into the receptor plant by genetic transformation method, and transformed offspring were identified, and the expression level of PEPC was analyzed from transformed progeny plants and transgenic plant protein, fat, fat composition changes were analyzed. In this study, from PEPC cloning, vector construction, genetic transformation, gene expression, composition analysis, the oil, protein and fatty acid composition changes were comprehensively and detail studied after PEPC gene inhibition the rapeseed, for the RNAi gene interference technologies and applied research to lay a theoretical basis. The main results include:1. PEPC gene sequence (Brassica napus.L) was searched in NCBI (D13987), and was blast conservative sequence among other sequences. The results showed that PEPC gene sequence was 6603bp, and a total of nine exons and eight introns and a polyA tail signal, located exon 4 of the 2637-2859 sequence of the strongest conservative. Using PROSITE database to predict PEPC gene function area, it found two activity sites in PEPC gene, and the first activity from the coding region is conserved sequences. Different types of rapeseed were PCR amplified to clon PEPC conservative sequence, then comparison revealed that the highly conserved consensus sequence from F008 (Brassica napus.L) in the cloned and published sequences of the NCBI, Brassica campestris.L and Brassica juncea.L, the conserved sequence similarity over 96%. Meanwhile, rapeseed storage protein promoter Napin was cloned by PCR amplification. It is the discovery of the promoter sequence of the original control,98% sequence similarity with the NCBI sequence of Napin, and gene transcription start functioning properly.2. Using Napin promoter and PEPC gene sequence (activity site sequence) from FOO8 (Brassica napus.L), RNA interference expression vector was constructed pCAMNapin-B1-NEI-B2, and Pbi35S-B1-NEI-B2 expression vector. In the vector construction process, the sequencing showed that replication plasmid. sequence was easily cut during the complementary sequence in E. coli, and cutting is random. By repeatedly connecting, cloning, ultimately, a experimental structure ihpRNA carrier was got consistent with the prediction, and the methylation sequencing to vector sequences showed that the carrier exists in two methylation sites, and methylation of the vector sequence prevent replication to be cut in E. coli. It analysis showed that the target fragment sequence does not methylated, and methylation sites is media vector, therefore, the vector still in normal transcription of receptor cells play double-stranded RNA role.3. By Agrobacterium-mediated Floral-dip and cotyledons, hypocotyls of genetic transformation methods, the plasmid expression vector was introduced into F008 and Westar(Brassica napus.L) receptor material, and those transgenic plants were identified by resistance screening, PCR amplification specificity, PCR-Southern, and DNA sequencing. Floral-dip and cotyledon, hypocotyl transformation methods were compared, and it result that Floral-dip transformation method more efficient, the average transformation efficiency reached 0.69%, less affected by genotype, and transformation process is simple, workable, without the need for tissue culture, but the resistance selection process cumbersome. The cotyledons, hypocotyls were lower transformation efficiency is 0.25% and 0.15%, respectively, influenced by the genotype, the complexity of operation, long cycle.4. With wild-type plants and transgenic plants compared to analysis by fluorescence quantitative PCR, it showed that PEPC gene expression level of transgenic plants was suppressed to some degrees, after 10 days flowering, the average inhibition rate of 58.82% in the transgenic F008, the average inhibition level was 61.5%, and the average inhibition rate reached 83.33%in the transgenic Westar, average 50% inhibitory level.25 days after flowering, PEPC gene expression inhibition rate of 23.53% on average in F008, the average inhibitory level of 29.75% and PEPC the average gene expression inhibition rate reached 33.33% in Westar, the average inhibition level was 58.5%. In T2 generation, the transgenic plants were amplified by PCR and methylation detection, and it result that in F008, T2 progeny was amplified material 187, the positive plant 128, the positive rate of 68.50%, and in Westar, T2 generation of plants were amplified by 144 positive plant 93, the positive rate of 64.58%, and positive plants were about 2/3, consistent with a pair of Mendelian genes, and Southern blot in T2 progeny analysis showed that transgenic plants are the single copy insertion, and the proportion of consistent PCR amplification. The methylation of PEPC gene was detected in transgenic plants and wild-type plants, found that methylation of transgenic plants was significantly increased, from 2 additional loci to increase ranging from 5 sites, indicating RNA interference to some extent on target gene expression regulation of DNA methylation, and it may be related to RNA interference of target gene expression regulation mechanisms.5. Transgenic plants and wild type of protein, oil and fatty acid composition analysis showed that T1 transgenic plants, an average points 2.41 percentage increase in oil content, relative to 5.97%, protein on average decreased 3.39 percentage points, the relative reduction 10.85% in F008, and in Westar average oil content of materials increased 1.51 percentage points, relative to 3.72%, protein decreased 3.42 percentage points on average,10.73% relative reduction. T2 transgenic plants, in F008 oil content of an average increase of 2.42 percentage points, relative to 5.18%, protein on average decreased 2.23 percentage points, the relative reduction 8.35%, and in Westar oil content of an average increase of 2.1 percentage points, relative to 5.16%, protein on average lower 1.34 percentage points, the relative reduction of 4.81%. Meanwhile, there are changes in the fatty acid composition in T2 generation of transgenic plants. In F008, oleic acid content than wild-type plants on average 4.30 percentage points higher, up 10.19 percentage points increase, linoleic acid decreased 2.89 percentage points on average, linolenic acid reduced by an average 1.20 percentage points, and in Westar oleic acid increased by an average 2.15 percentage points, the maximum increase 5.87 percent,2.04 percentage points lower linoleic acid, linolenic acid reduced by 0.11 percentage points.