反義4CL基因調(diào)控?zé)煵菽举|(zhì)素合成的研究
發(fā)布時間：2019-01-05 20:20
【摘要】：木質(zhì)素存在于植物細(xì)胞的細(xì)胞壁中,與纖維素和半纖維素以甲苯醚鍵的形式連接在一起,木質(zhì)素單體之間也靠穩(wěn)定的化學(xué)鍵相連接。木質(zhì)素這種存在方式妨礙了纖維素和半纖維素的消化和利用。4-香豆酸輔酶A連接酶(4CL)在木質(zhì)素單體合成過程中催化羥基肉桂酸生成羥基肉桂酰輔酶A,是連接苯丙烷途徑和木質(zhì)素合成特異途徑的關(guān)鍵酶。研究表明,抑制4CL基因的表達(dá)能夠有效的降低植物木質(zhì)素含量。本實驗將反義4CL基因通過農(nóng)桿菌介導(dǎo)的方法轉(zhuǎn)入模式植物煙草中,通過降低煙草4CL基因的表達(dá),達(dá)到降低煙草木質(zhì)素含量的目的。本實驗研究取得以下進展：1)將含有反義4CL基因的表達(dá)載體通過農(nóng)桿菌介導(dǎo)的方法轉(zhuǎn)入模式植物煙草中,獲得TO代陽性煙草轉(zhuǎn)化植株。TO代植株白花授粉,收獲種子,種植后獲得T1代植株。PCR、RT-PCR檢測、目的片段測序的結(jié)果表明,反義4CL基因已經(jīng)穩(wěn)定遺傳到T1代中,并且正常表達(dá)。2)對T1代煙草植株進行遺傳穩(wěn)定檢測,共檢測100棵T1代煙草植株,陽性植株和陰性植株的比例為77：23,接近于3：1,表明反義4CL基因在T1代煙草植株的遺傳符合孟德爾遺傳定律。3)對T1代煙草進行qRT-PCR檢測,觀察T1代煙草4CL1基因表達(dá)情況,結(jié)果表明：檢測的兩株T1代煙草轉(zhuǎn)化植株葉片4CL1表達(dá)量分別降低了21.58%和17.43%。對T1代煙草的4CL酶含量進行ELISA檢測,結(jié)果表明,檢測的兩株T1代植株莖4CL酶含量分別降低了11.19%和9.8%,說明該反義基因能夠干擾煙草4CL基因的表達(dá)。4)對T1代轉(zhuǎn)基因煙草進行生長指標(biāo)和生理生化指標(biāo)檢測,結(jié)果表明：T1代煙草莖木質(zhì)素含量下降了11.87%,根木質(zhì)素含量降低了14.23%。轉(zhuǎn)基因植株與野生型植株生長一致,在株高、株重等方面差異并不明顯。說明反義4CL基因的轉(zhuǎn)入在降低了煙草木質(zhì)素含量的同時并沒有影響煙草的正常生長。
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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2 武小芬;蘇小軍;陳亮;陳靜萍;王克勤;;木質(zhì)素結(jié)構(gòu)分析方法研究進展[J];可再生能源;2015年02期
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