【作者】 陈伟；

【导师】 刘立侠；

【作者基本信息】 东北师范大学 ， 植物学， 2011， 博士

【摘要】 应用基因工程技术调节大豆脂类代谢途径,探索相关基因的调控机制,对于大豆脂肪酸合成的基础研究和品质育种是一项有效和快捷的途径。本论文以吉林大豆为材料,以蛋白质和脂肪酸代谢的“底物竞争”假说为依据,采用基因工程技术,探讨大豆fad2(脂肪酸脱氢酶)基因和pepc(磷酸烯醇式丙酮酸羧化酶)基因在调控大豆种子脂肪酸合成代谢中的作用,进而选育高油酸和高油的转基因大豆材料。根据基因沉默原理,克隆了大豆fad2和pepc基因的片段,分别构建了基因沉默ihpRNA高效表达载体pBIGFP-FAD2、pBI-FAD2和pBI-PEP;以农杆菌介导的遗传转化方法并结合嫁接技术转化和筛选大豆吉林35,对再生植株进行分子鉴定,基因功能检测和一系列生理生化实验,分析和研究了pepc基因和acc基因在大豆种子发育过程中的时间表达模式。通过农杆菌介导的遗传转化方法将pBIGFP-FAD2载体转化三个高油大豆品种吉林35,吉林47和吉科豆1号。检测报告基因GFP的表达,结果表明,吉林35是大豆胚尖再生系统中最敏感的品种,其表达效率可达到42.9%。本实验优化了大豆胚尖转化系统的卡那霉素筛选压力,并首次尝试将嫁接技术应用到大豆再生系统中替代生根炼苗过程,再生苗的成活率从10%提高到80%,炼苗周期由45天缩短到10天。pBI-FAD2转化吉林35共获得180株再生植株,其中34株PCR阳性植株经分子检测证明外源基因已经整合到植物基因组中,8份材料种子油酸含量有不同程度的提高。阳性材料通过Southern和Northern杂交检测、脂肪酸含量及油酸脱氢比例(ODP)分析,证明ihpRNA沉默fad2基因,导致了油酸含量增加、亚油酸含量降低的效果,并且具有孟德尔单因素遗传规律。阳性植株种子脂肪酸检测表明,油酸含量从对照的17.5%提高到50%左右。通过农杆菌介导转化pBI-PEP载体到吉林35大豆中,共获得再生植株265株,PCR阳性植株64株,Southern杂交证明目的基因整合至基因组中。75%阳性植株(48株)的油含量有不同程度的提高,其中9份阳性材料T1-T4代大豆油含量平均为22.55%,比对照(20.75%)提高了1.8%个百分点;蛋白质含量平均为34.07%,比对照(36.77%)下降2.7%。选取其中4份材料,检测阳性材料(T3代种子)的脂肪酸组分表明,高油材料与对照差异很小。在种子的五个不同发育期检测这4份材料的PEPC酶活性,结果表明其PEPC酶活均较对照低。使用Real-timePCR检测4份种子不同发育期pepc基因和acc基因的表达,结果表明不同发育期阳性种子pepc基因的表达量均比对照低,是同时期对照表达量的0.9倍、0.78倍、0.56倍、0.15倍和0.03倍。在种子不同发育期,阳性材料acc基因的表达量均比同时期对照高,分别是同时期对照表达量的1.23倍、1.56倍、3.01倍、1.72倍和18.78倍。转化阳性材料通过Southern杂交、pepc基因和acc基因的荧光定量表达、脂肪含量、蛋白质含量及酶活的分析,证明ihpRNA沉默pepc基因达到了油脂含量提高,蛋白质含量下降的效果,为验证植物脂肪酸代谢的“底物竞争”假说提供了新的证据。总之,本研究将含有fad2基因和pepc基因的沉默表达载体分别转化大豆,在转基因材料中获得了较好的fad2基因和pepc基因的沉默效果,并筛选出高油酸和高油大豆材料。为深入研究基因工程调控大豆油脂代谢和培育高油、高油酸大豆等方面提供了一定的基础和依据。同时在大豆遗传转化中利用嫁接技术,是提高胚尖转化系统再生频率的有效手段,为大豆再生系统的优化提供了新方法。更多还原

【Abstract】 It is an effective and efficient way for the study of soybean fatty acid synthesisand quality breeding using soybean genetic engineering regulation of lipid metabolicpathways and to explore mechanisms of gene regulation. In this paper, Jilin soybeanswere chosen as materials, base on protein and fatty acid metabolism of the "substratecompetition" hypothesis and using genetic engineering techniques to explore roles ofsoybean fad2 (fatty acid desaturase) gene and pepc (phosphoenolpyruvate carboxylase)gene in the regulations of soybean seeds fatty acid synthesis metabolism, and thenselect high-oleic and high oil breedings of genetically modified soybean. In this study,gene silencing vectors pBIGFP-FAD2、pBI-FAD2 and pBI-PEP were constructedaccording to gene silencing principle in plant, which contained fatty acid metabolismgenes fad2 (oleic acid dehydrogenase) and pepc (Phosphoenolpyruvate carboxylase),They were transformed into soybeans to regulate fatty acid matabolizm withenhancing fatty acids synthesis and oleic acid accumulation, and then, to screen outhigh oleic and high oil soybean materials.The pBIGFP-FAD2 vector was transformed into Jilin 35, Jilin 47 and Jike 1 usingAgrobacterium tumefaciens mediated method espectively, which were three high-oilcultivars in Jilin Province. The expression of GFP showed that, Jilin 35 is the mostsensitive cultivar in embryonic tip regeneration system. Transformation efficiency wasup to 42.9%. In this study, we optimized kanamycin concentration in embryonic tipregeneration system and attempted to applicate graft technology instead of thetraditional rooting method in soybean regeneration system for the first time. Graftingmethod survival rate can be up to 80% compare to 10% in root acclimatization method,the cycle was deduced from 45 days to 10 days. Total 180 regenerated plants wereobtained in the pBI-FAD2 transformation Jilin 35 soybean study. Molecular detectionshowed that the exogenous DNAs intergrated in 34 transgenic positive plantsgenomes. Southern hybridization, Northern hybridization, fatty acid contents and oleicacid proportion (ODP) assays indicated that fad2 gene expression was inhibited by theihpRNA construct, which consistant with high oleic acid and low linoleic acid in theseeds and a single-gene segregation pattern of Mendel. The fatty acid analysis of thetransgenic positive seeds showed that oleic acid content increased from 17.5% to 41-52%. Expression vector pBI-PEP was transformed into Jilin 35 soybeans by A.tumefaciens method. Total 265 regenerated plants and 64 PCR-positive transgenicplants were obtained. Southern hybridization showed that targeted genes wereintegrated into the plants genomes. 48 regenerated plants with different degrees of oilcontent, number of them accounted for 75% in the positive transformation materials.Nine materials of these soybeans had 22.55% oil content in T1-T4 on average, 1.8%higher than the control (20.75%). The average protein content of these 9 positivematerials was 34.07%, down 2.7% compared with the control (36.77%). Four seedpositive materials were chosen to analyze fatty acid composition in T3 showed littledifference between the controls. At five different developing stages, four positivematerials were detected PEPC activity. The result showed PEPC activitys of positivematerials at different stages were lower than that of control respectively. Expressionsof pepc and acc in developing seeds assay using Real-time PCR showed that, pepcexpressions at all detected stages were lower than the expressions of the control at thesame stage respectively, the expressions of them were 0.9 times, 0.78 times and 0.56times, 0.15 times and 0.03 times of the controls. Acc expressions of transgenicmaterials at all stage are larger than the control respectively, the expressions of themwere 1.23 times, 1.56 times and 3.01 times, 1.72 times and 18.78 times of the controls.The positive materials were assayed by Southern blot, fluorescence quantitative geneexpressions of pepc and acc, fat composition, protein content and enzyme activityanalysis, the results confirmed ihpRNA silence pepc genetically improved oil contentand decreased protein content with fatty acid metabolism in plants, it provided newevidence to verify the "substrate competition" hypothesis.Overall, we constructed two silence vectors contained fad2 and pepc andtransformed them into soybeans. We obtained better silencing effect on fad2 and pepcexpression then had screened high oleic acid and high oil soybean materials. Itprovided a foundation and basis materials to study lipid metabolism and breeding highoil, high oleic acid soybeans. For the first time we showed the graft could be apply inembryonic tip regeneration system in soybean seedlings. Graft improves the frequencyof embryonic tips regeneration system. It is provides a new approach for optimizatingof soybean regeneration system.更多还原