Genetic Analysis Of High Oleic In The Transgenic Brassica Napus Line W-4and The Sequence Analysis Of Its T-DNA Integration Sites

Oleic(18:1) acid is an important monounsaturated fatty acids, it is the high oleic acid that can reduce LDL cholesterol levels, slow down atherosclerosis and prevent the occurrence of coronary artery disease Effectively; it can be used for the production of bio-diesel because of methyling, therefore the nutritional and economic value of rapeseed oil is determined by content of oleic acid. The material in this research is obtained by applying ihpRNA induced fad2gene silencing. IhpRNA induced gene silene expression、 inheritance of oleic acid content, W-4transformation event was study in this research.The main results were as follows:1.Inheritance of oleic acid content was investigated in P1、P2、F1、B1、B2、F2and P1、 P2、RF1、RB1、RB2、RF2generations derived from the cross of "W-4"×"westar". The results showed that:(1)The oleic acid content of the transgenic lines W-4of the T2、T3、 T4、T5generations were more than80%indicating that the oleic acid content in W-4seeds is stability between the various generationstraits.(2)No partial and cytoplasmic effects for oleic acid concentration were observed in the analysis of F1seeds and RF1seeds, respectively from reciprocal crosses. Oleic acid concentration of F2%RF2seeds segregated following a3:1ratio, suggesting dominant monogenic inheritance. This was confirmed in the B2and RB2to "westar", which segregated following1:1ratio.(3) The Kan-resistance test results also show the same inheritance law.2.The relative expression quantitative of fad2gene during in seed development and in the vegetable organs such as root, stem, leaf in winter seedling stages of both the transgenic line W-4and westar by the quantitative real-time PCR. Results demonstrated that:(1)the fad2gene relative expression levels were gradually increasing in the seeds of the7th DAF to28th DAF of the westar. But in the seeds of transgenic line W-4the relative quantity of fad2gene expression began decreasing significantly since the21th DAF compared with westar. The fad2gene expression is consistent between W-4and westar in vegetable organs. The analysis of the fatty acid composition showed a higher oleic acid content and lower polyunsaturated fatty acid in seeds of transgenic line, but no significant difference in the vegetable organs. The results above validated that RNA interference in transgenic line W-4is of seed-specific expression, without interfering with the fad2gene expression in other organs. The feature is of particular significance for the biosafety assessment of genetically modified rapeseed.3. The flanking sequence to both the right border and left border of the T-DNA in the genome was obtained by Tail-PCR. The results showed that:(1) he right border flanking sequence is470bp which includes a290bp genome sequence and a180bp vector sequence based on VecScreen. Further sequence alignment analysis showed that the180bp was identical to the RB border of pCNFIRnos but with a62bp deletion. The left border flanking sequence is641bp including a365bp genome sequence and a276bpvector sequence. The276bp was identical to the left border of pCNFIRnos except a change of G to A. The right border base is consisted of32.6%GC and67.4%AT; the left border base includes31.27%GC and68.73%AT.The results show that T-DNA integrates in the AT-rich region.(3)The specificity of the established methods were tested well after testing with other transgenic rapeseed, and detection sensitivity of them were0.05%. The event-specific detection method has a high degree of specificity and good sensitivity for the genetically modified rapeseed lines W-4identification.