反义4CL基因调控烟草木质素合成的研究

发布时间：2019-01-05 20:20

【摘要】：木质素存在于植物细胞的细胞壁中,与纤维素和半纤维素以甲苯醚键的形式连接在一起,木质素单体之间也靠稳定的化学键相连接。木质素这种存在方式妨碍了纤维素和半纤维素的消化和利用。4-香豆酸辅酶A连接酶(4CL)在木质素单体合成过程中催化羟基肉桂酸生成羟基肉桂酰辅酶A,是连接苯丙烷途径和木质素合成特异途径的关键酶。研究表明,抑制4CL基因的表达能够有效的降低植物木质素含量。本实验将反义4CL基因通过农杆菌介导的方法转入模式植物烟草中,通过降低烟草4CL基因的表达,达到降低烟草木质素含量的目的。本实验研究取得以下进展：1)将含有反义4CL基因的表达载体通过农杆菌介导的方法转入模式植物烟草中,获得TO代阳性烟草转化植株。TO代植株白花授粉,收获种子,种植后获得T1代植株。PCR、RT-PCR检测、目的片段测序的结果表明,反义4CL基因已经稳定遗传到T1代中,并且正常表达。2)对T1代烟草植株进行遗传稳定检测,共检测100棵T1代烟草植株,阳性植株和阴性植株的比例为77：23,接近于3：1,表明反义4CL基因在T1代烟草植株的遗传符合孟德尔遗传定律。3)对T1代烟草进行qRT-PCR检测,观察T1代烟草4CL1基因表达情况,结果表明：检测的两株T1代烟草转化植株叶片4CL1表达量分别降低了21.58%和17.43%。对T1代烟草的4CL酶含量进行ELISA检测,结果表明,检测的两株T1代植株茎4CL酶含量分别降低了11.19%和9.8%,说明该反义基因能够干扰烟草4CL基因的表达。4)对T1代转基因烟草进行生长指标和生理生化指标检测,结果表明：T1代烟草茎木质素含量下降了11.87%,根木质素含量降低了14.23%。转基因植株与野生型植株生长一致,在株高、株重等方面差异并不明显。说明反义4CL基因的转入在降低了烟草木质素含量的同时并没有影响烟草的正常生长。
[Abstract]:Lignin exists in the cell wall of plant cells and is linked to cellulose and hemicellulose in the form of diphenyl ether bonds. Lignin monomers are also linked by stable chemical bonds. The existence of lignin hinders the digestion and utilization of cellulose and hemicellulose. 4-coumaric acid coenzyme A ligase (4CL) catalyzes hydroxycinnamic acid to produce hydroxycinnamyl coenzyme A during lignin monomer synthesis. It is a key enzyme linking phenylpropane pathway and lignin synthesis pathway. The study shows that inhibiting the expression of 4CL gene can effectively reduce the content of lignin in plants. In this study, antisense 4CL gene was transferred into model plant tobacco by Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens, and the content of lignin was reduced by reducing the expression of 4CL gene in tobacco. The main results of this study are as follows: 1) the expression vector containing antisense 4CL gene was transferred into the model plant tobacco by Agrobacterium tumefaciens to obtain the TO generation positive tobacco transformation plants. The TO generation plants were pollinated in white flowers and harvested seeds. The results of target fragment sequencing showed that antisense 4CL gene had been stably inherited to T1 generation and expressed normally. 2) genetic stability of T1 generation tobacco plants was detected. A total of 100 T1 tobacco plants were tested. The ratio of positive to negative plants was 77: 23, which was close to 3: 1. The results showed that the inheritance of antisense 4CL gene in T1 generation tobacco plants was in accordance with Mendelian's law of heredity. 3) qRT-PCR detection of T1 generation tobacco was carried out, and the expression of 4CL1 gene in T1 generation tobacco was observed. The results showed that the 4CL1 expression levels in the leaves of the two T1 generation tobacco transformed plants were reduced by 21.58% and 17.43%, respectively. The content of 4CL enzyme in T1 generation tobacco was detected by ELISA. The results showed that the content of 4CL enzyme in stem of two T1 generation plants was decreased by 11.19% and 9.8%, respectively. The results showed that the antisense gene could interfere with the expression of 4CL gene in tobacco. 4) the growth index and physiological and biochemical indexes of T1 generation transgenic tobacco were detected. The results showed that the lignin content of T1 generation tobacco stem decreased by 11.87%. The lignin content of root decreased 14.23%. The growth of transgenic plants was consistent with that of wild-type plants, but there were no significant differences in plant height and plant weight between transgenic plants and wild-type plants. The results showed that the antisense 4CL gene did not affect the normal growth of tobacco while reducing the content of lignin.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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