AtSUS3基因的克隆及對(duì)棉花的遺傳轉(zhuǎn)化研究
發(fā)布時(shí)間：2018-07-18 13:02
【摘要】：棉花,錦葵科棉屬植物,是世界上主要的經(jīng)濟(jì)作物之一。棉纖維由胚珠部分表皮細(xì)胞發(fā)育形成,涉及許多基因的表達(dá)調(diào)控。UDPG(uridine 5'-diphosphoglucose)是纖維素合成的底物,蔗糖合成酶(Sucrose Synthase, SuSy)可作用于表皮細(xì)胞產(chǎn)生UDPG,同時(shí)降低細(xì)胞中UDP (uridine diphosphate)的含量,提高細(xì)胞膨壓,促進(jìn)細(xì)胞伸長(zhǎng)及纖維素合成。棉花無(wú)絨毛突變體和野生型0 DPA (day post anthesis)的胚珠比較,表皮細(xì)胞突起銳減,同時(shí)未檢測(cè)到SuSy的轉(zhuǎn)錄及酶活性。棉花中蔗糖合成酶3(SUS3)基因的低表達(dá),會(huì)導(dǎo)致0 DPA胚珠表面突起減少,3 DPA的棉纖維變短。本實(shí)驗(yàn)室前期工作發(fā)現(xiàn)干涉擬南芥AtSUS3基因的表達(dá),引起其它SUS成員代償性增加,進(jìn)而促進(jìn)轉(zhuǎn)基因擬南芥抽苔、果實(shí)成熟,其角果產(chǎn)率也均高于野生型植株。在此基礎(chǔ)上,本研究克隆了AtSUS3基因,并將此基因連接植物表達(dá)載體pBI121,轉(zhuǎn)化農(nóng)桿菌LBA4404,利用兩種方法對(duì)棉花進(jìn)行遺傳轉(zhuǎn)化,以期獲得轉(zhuǎn)AtSUS3棉。已取得的結(jié)果如下：1.克隆AtSUS3基因。從擬南芥中克隆AtSUS3,該基因cDNA全長(zhǎng)2430 bp,編碼809個(gè)氨基酸,AtSUS3的分子量為92.00 kDa,理論等電點(diǎn)為5.85,疏水性平均值為-0.276,含量最豐富的氨基酸為L(zhǎng)eu,Glu,Val,Gly。蛋白結(jié)構(gòu)預(yù)測(cè)分析表明AtSUS3蛋白屬于糖基轉(zhuǎn)移酶家族,含有2個(gè)功能域,即N端的蔗糖合成結(jié)構(gòu)域和C端的糖基轉(zhuǎn)移結(jié)構(gòu)域。在AtSUS3的二級(jí)結(jié)構(gòu)中,α-螺旋占34.49%,隨機(jī)卷曲占48.95%,延伸鏈占16.56%。其中,a-螺旋和隨機(jī)卷曲是AtSUS3最主要的結(jié)構(gòu)元件。2.構(gòu)建表達(dá)載體。將AtSUS3基因連入表達(dá)載體pBI121,命名為:pBI121-AtSUS3,并轉(zhuǎn)入農(nóng)桿菌LBA4404中。利用農(nóng)桿菌介導(dǎo)棉花下胚軸轉(zhuǎn)化法對(duì)棉花進(jìn)行轉(zhuǎn)化。選取飽滿、脫絨的棉種無(wú)菌培養(yǎng),將生長(zhǎng)7天后的棉花子葉下胚軸切段,通過(guò)農(nóng)桿菌侵染3分鐘,漂洗后共培養(yǎng)48小時(shí),在抗性培養(yǎng)基上誘導(dǎo)形成愈傷組織,再進(jìn)一步誘導(dǎo)形成胚性愈傷組織,通過(guò)胚胎發(fā)生等過(guò)程獲得再生植株。3.分析嫁接方法。分別對(duì)劈接法,合接法,貼接法等方法進(jìn)行分析,結(jié)果表明：嫁接土培苗時(shí)合接法成活率最高,劈接法嫁接后棉苗生長(zhǎng)最好。無(wú)菌苗嫁接中,劈接法成活率最高,棉苗生長(zhǎng)良好。4.獲得轉(zhuǎn)AtSUS3棉花。轉(zhuǎn)AtSUS3基因的再生植株通過(guò)劈接法嫁接獲得19株轉(zhuǎn)AtSUS3棉花。通過(guò)ELISA方法研究了轉(zhuǎn)基因棉花中蔗糖合成酶(SuSy),蔗糖轉(zhuǎn)化酶(INV),蔗糖磷酸合成酶(SPS)的含量及酶活性,結(jié)果表明：轉(zhuǎn)基因棉花普遍高于野生型棉花,但各陽(yáng)性株系間存在差異。5.探索棉花活體胚轉(zhuǎn)化外源基因的方法。用含外源基因GFP的農(nóng)桿菌侵染去掉半個(gè)子葉的棉花種子胚,農(nóng)桿菌OD600為0.4-0.6,侵染3小時(shí),共培養(yǎng)48小時(shí),用含100 mg/L卡那霉素的MS基礎(chǔ)培養(yǎng)基篩選,生長(zhǎng)30天后的棉苗在卡那霉素敏感性實(shí)驗(yàn)具有抗性,在DNA水平,轉(zhuǎn)錄水平上均可檢測(cè)到GFP,棉花葉片也可檢測(cè)到綠色熒光,但40天后外源基因表達(dá)減弱,60天后丟失,表明該轉(zhuǎn)基因植物為嵌合體。
[Abstract]:Cotton is one of the major cash crops in the world. Cotton fiber is formed by ovule partial epidermal cells. UDPG (uridine 5'-diphosphoglucose) is the substrate of cellulose synthesis. Sucrose Synthase (SuSy) can act on the epidermal cells to produce UDPGs and decrease the content of (uridine diphosphate) in the cells. Increase cell pressure, promote cell elongation and cellulose synthesis. Compared with the ovule of wild-type 0DPA (day post anthesis), the epidermal processes of cotton without villous mutants decreased sharply, and the transcription and enzyme activity of SuSy were not detected. The low expression of sucrose synthase 3 (SUS3) gene in cotton resulted in the reduction of cotton fiber from the surface of 0 DPA ovules. Our previous work found that interfering with the expression of AtSUS3 gene in Arabidopsis thaliana caused compensatory increase of other SUS members, and then promoted transgenic Arabidopsis thaliana to sprout coating and mature fruit, and the rate of keratin production was also higher than that of wild-type plants. On this basis, AtSUS3 gene was cloned and ligated into plant expression vector pBI121to transform Agrobacterium tumefaciens LBA4404. Two methods were used to carry out genetic transformation of cotton in order to obtain transgenic cotton. The results achieved are as follows: 1. AtSUS3 gene was cloned. AtSUS3 was cloned from Arabidopsis thaliana. The total length of AtSUS3 gene was 2430 BP, encoding 809 amino acids, the molecular weight of AtSUS3 was 92.00 kDa, the theoretical isoelectric point was 5.85, and the hydrophobic average was -0.276. Protein structure prediction analysis showed that AtSUS3 protein belongs to the glycosyltransferase family and contains two functional domains: N-terminal sucrose biosynthesis domain and C-terminal glycosyltransferase domain. In the secondary structure of AtSUS3, 偽 -helix is 34.49, random crimp is 48.95 and extension chain is 16.56. The main structural components of AtSUS3, I. e., A helix and random crimp, are. 2. 2. Construct expression vector. AtSUS3 gene was inserted into the expression vector pBI121and named as: pBI121-AtSUS3, and transferred into Agrobacterium tumefaciens LBA4404. Cotton was transformed by Agrobacterium tumefaciens mediated Hypocotyl transformation. After 7 days of growth, cotton cotyledon Hypocotyl was cut into segments, infected with Agrobacterium tumefaciens for 3 minutes, then rinsed for 48 hours, then induced to form callus on resistant medium. Embryogenic calli were further induced and regenerated plants were obtained by embryogenesis. The grafting method was analyzed. The results showed that the survival rate of the grafting method was the highest, and the cotton seedling growth was the best after the split grafting method. In the grafting of sterile seedlings, the survival rate of split grafting was the highest, and cotton seedlings grew well. 4. The cotton was transferred to AtSUS3. Nineteen transgenic plants of AtSUS3 were obtained by split-grafting. The content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were studied by Elisa. The results showed that the content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were generally higher than those in wild type cotton. To explore the method of transformation of foreign gene from cotton in vivo embryo. Agrobacterium tumefaciens containing foreign gene GFP was used to infect and remove half cotyledon of cotton seed embryo. Agrobacterium-OD600 was 0.4-0.6, infected for 3 hours, co-cultured for 48 hours, and screened by MS basic medium containing 100 mg / L kanamycin. After 30 days of growth, cotton seedlings were resistant to kanamycin, GFP could be detected at DNA level and transcription level, green fluorescence could also be detected in cotton leaves, but after 40 days, the expression of exogenous genes was decreased and lost after 60 days. The results showed that the transgenic plants were chimerism.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2;S562
本文編號(hào)：2131981
[Abstract]:Cotton is one of the major cash crops in the world. Cotton fiber is formed by ovule partial epidermal cells. UDPG (uridine 5'-diphosphoglucose) is the substrate of cellulose synthesis. Sucrose Synthase (SuSy) can act on the epidermal cells to produce UDPGs and decrease the content of (uridine diphosphate) in the cells. Increase cell pressure, promote cell elongation and cellulose synthesis. Compared with the ovule of wild-type 0DPA (day post anthesis), the epidermal processes of cotton without villous mutants decreased sharply, and the transcription and enzyme activity of SuSy were not detected. The low expression of sucrose synthase 3 (SUS3) gene in cotton resulted in the reduction of cotton fiber from the surface of 0 DPA ovules. Our previous work found that interfering with the expression of AtSUS3 gene in Arabidopsis thaliana caused compensatory increase of other SUS members, and then promoted transgenic Arabidopsis thaliana to sprout coating and mature fruit, and the rate of keratin production was also higher than that of wild-type plants. On this basis, AtSUS3 gene was cloned and ligated into plant expression vector pBI121to transform Agrobacterium tumefaciens LBA4404. Two methods were used to carry out genetic transformation of cotton in order to obtain transgenic cotton. The results achieved are as follows: 1. AtSUS3 gene was cloned. AtSUS3 was cloned from Arabidopsis thaliana. The total length of AtSUS3 gene was 2430 BP, encoding 809 amino acids, the molecular weight of AtSUS3 was 92.00 kDa, the theoretical isoelectric point was 5.85, and the hydrophobic average was -0.276. Protein structure prediction analysis showed that AtSUS3 protein belongs to the glycosyltransferase family and contains two functional domains: N-terminal sucrose biosynthesis domain and C-terminal glycosyltransferase domain. In the secondary structure of AtSUS3, 偽 -helix is 34.49, random crimp is 48.95 and extension chain is 16.56. The main structural components of AtSUS3, I. e., A helix and random crimp, are. 2. 2. Construct expression vector. AtSUS3 gene was inserted into the expression vector pBI121and named as: pBI121-AtSUS3, and transferred into Agrobacterium tumefaciens LBA4404. Cotton was transformed by Agrobacterium tumefaciens mediated Hypocotyl transformation. After 7 days of growth, cotton cotyledon Hypocotyl was cut into segments, infected with Agrobacterium tumefaciens for 3 minutes, then rinsed for 48 hours, then induced to form callus on resistant medium. Embryogenic calli were further induced and regenerated plants were obtained by embryogenesis. The grafting method was analyzed. The results showed that the survival rate of the grafting method was the highest, and the cotton seedling growth was the best after the split grafting method. In the grafting of sterile seedlings, the survival rate of split grafting was the highest, and cotton seedlings grew well. 4. The cotton was transferred to AtSUS3. Nineteen transgenic plants of AtSUS3 were obtained by split-grafting. The content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were studied by Elisa. The results showed that the content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were generally higher than those in wild type cotton. To explore the method of transformation of foreign gene from cotton in vivo embryo. Agrobacterium tumefaciens containing foreign gene GFP was used to infect and remove half cotyledon of cotton seed embryo. Agrobacterium-OD600 was 0.4-0.6, infected for 3 hours, co-cultured for 48 hours, and screened by MS basic medium containing 100 mg / L kanamycin. After 30 days of growth, cotton seedlings were resistant to kanamycin, GFP could be detected at DNA level and transcription level, green fluorescence could also be detected in cotton leaves, but after 40 days, the expression of exogenous genes was decreased and lost after 60 days. The results showed that the transgenic plants were chimerism.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2;S562
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 蔡云巧;AtSUS3基因的克隆及對(duì)棉花的遺傳轉(zhuǎn)化研究[D];陜西師范大學(xué);2016年
，本文編號(hào)：2131981
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