小麦近缘属植物果聚糖合成相关基因的克隆和功能验证及小麦共转化体系的优化
发布时间：2018-08-12 11:23
【摘要】：非生物胁迫是影响作物生长发育、限制其产量的重要环境因子,作物如何应对非生物胁迫是世界性的研究课题。果聚糖的积累有利于增强植物对逆境胁迫的抵抗能力。小麦近缘属植物具有抗旱、抗寒、抗盐及耐瘠薄等优良特性。因此,克隆其果聚糖合成相关基因,鉴定其功能并进行小麦抗逆育种改良研究具有重要的理论和现实意义。转基因植物的载体骨架序列及抗性标记是转基因生物安全性争论的焦点,是限制转基因植物商业化的重要因素。基因枪介导的最小表达盒(目的基因和筛选基因)按一定摩尔比共转化受体材料可能是有效去除载体序列和标记基因的一种高效的遗传转化法。本研究以小麦近缘属植物华山新麦草(Psathyrostachys huashanica Keng)、簇毛麦(Dasypyrum villosum(L.)),大赖草(Leymus racemosus)、滨麦(Leymus mollis)为材料,利用genome walking结合RT-PCR技术克隆到华山新麦草和簇毛麦果聚糖-6-果糖基转移酶基因(6-SFT);利用RT-PCR结合RACE技术克隆到大赖草和滨麦6-SFT基因及华山新麦草、簇毛麦、大赖草果聚糖-1-果糖基转移酶基因(1-FFT),华山新麦草蔗糖-1-果糖基转移酶基因(1-SST),并对其进行基因结构和序列分析;以p1300-35SN为载体,构建植物表达载体p1300-35SN-FBEs。通过农杆菌介导法转化烟草,对转基因植株进行抗旱、抗寒和抗盐性鉴定,并测定与果聚糖含量相关的重要生理指标。以质粒pAHC25为参照,将质粒pAHC25和pEasy-Blunt-bar所对应的gus和bar基因的线性表达盒通过酶切消化并纯化回收,将bar或pAHC20与gus按摩尔比1:1、1:2、1:3平行地共转化普通小麦科农199幼胚,以期确定筛选基因与目标基因的最佳摩尔比,建立安全高效的基因枪介导的普通小麦表达盒共转化体系。本文得到的主要研究结果如下:1.分离到多种果聚糖合成酶基因:克隆到来自华山新麦草、簇毛麦、大赖草和滨麦的4个6-SFT基因,分别命名为Ph-6-SFT(1851 bp)、Dv-6-SFT(1863 bp)、Lr-6-SFT(1863 bp)和Lm-6-SFT(1866 bp);克隆到华山新麦草、簇毛麦、大赖草的3个1-FFT基因,分别命名为Ph-1-FFT(1989 bp)、Dv-1-FFT(1950 bp)和Lr-1-FFT(1989 bp);克隆到华山新麦草的1个1-SST基因,命名为Ph-1-SST(2001 bp);2.完成多个果聚糖果糖基转移酶基因载体构建:将Ph-1-SST、Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因分别插入到p1300-35SN载体获得7个基因的植物表达载体;3.果聚糖合成酶基因转化烟草及转基因植株的分子鉴定:将所构建的植物表达载体p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT/Ph-6-SFT/Dv-6-SFT/Lr-6-SFT通过农杆菌介导法转化烟草,经潮霉素筛选后每个载体分别获得113、98、101、65、69、72共518株转基因烟草植株,应用基因特异性引物对6个载体的转基因烟草植株进行基因组PCR、RT-PCR鉴定,每个载体分别获得64、53、55、24、38、43共277株转基因阳性植株。4.转基因植株抗逆性鉴定:表型分析结果表明,这些基因的表达都能够增强转基因烟草对旱、冷、盐胁迫的耐受能力。不同物种的6-SFT基因比1-FFT基因抗逆效果好,不同物种的同一基因抗逆性无明显差异。相比之下,Ph-6-SFT和Dv-6-SFT基因的抗逆效果较好。5.转基因植株生理指标的测定:生理指标测定结果表明,在旱及冷胁迫处理条件下,与转空载体的对照植株相比转Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因的阳性植株具有较高的果聚糖、可溶性糖及脯氨酸含量;较低的丙二醛(Malondialdehyde,MDA)含量。旱或冷胁迫处理后的转基因植株果聚糖、可溶性糖和脯氨酸含量显著高于正常条件下转基因植株的果聚糖、可溶性糖和脯氨酸含量。旱和冷胁迫后转基因植株丙二醛含量增加不显著,而对照植株丙二醛含量显著增加。这些结果表明Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因的表达均能显著增强转基因植株果聚糖等渗透调节物质的积累从而使转基因植株能更好的抵抗旱和冷胁迫所致的氧化损伤。转不同基因的转基因植株各生理指标的比较结果表明,与1-FFT基因相比6-SFT基因对提高作物逆境胁迫抗性更有效。不同物种的同一基因各生理指标之间的差异不显著。相比之下,Dv-6-SFT和Ph-6-SFT基因对提高植物的抗旱、抗寒效果较好。以上试验结果表明Dv-6-SFT和Ph-6-SFT基因可应用到作物抗逆育种中。6.基因枪介导的最小表达盒共转化小麦最佳摩尔比的确立:基因枪介导的完整质粒或表达盒共转化普通小麦,除草剂草丁膦(phosphinothricin,PPT)筛选后共获得3964株T0代小麦抗性植株,PCR鉴定共获得70株推定的GUS阳性植株,GUS染色检测共获得56株阳性植株。转化效率统计结果显示,获得完整质粒的转基因植株11株,转化率为0.70%;gus+bar表达盒的转基因植株30株,转化率为0.16-1.03%;gus+pAHC20的转基因植株29株,转化率为0.09-0.98%。其中pAHC20/bar:gus摩尔比为1:2时转化率显著高于其他组合。Southern blot分析结果表明,gus基因已成功整合到小麦基因组中。表达盒共转化小麦最佳摩尔比的确立为普通小麦遗传转化果聚糖相关基因提供参考依据。7.将植物表达载体p1301-Ph-6-SFT/Dv-6-SFT通过基因枪介导的最小表达盒共转化法转化普通小麦科农199,对T0代植株进行抗性筛选、叶片GUS染色、gus基因的PCR检测,结果表明已成功将表达载体转入小麦基因组,并且确认各获得1株转基因阳性株系。
[Abstract]:Abiotic stress is an important environmental factor affecting the growth and development of crops and limiting their yield. How crops respond to abiotic stress is a worldwide research topic. Fructan accumulation is conducive to enhancing the resistance of plants to stress. It is of great theoretical and practical significance to identify the genes involved in fructan biosynthesis, identify their functions and study the improvement of wheat stress resistance breeding. In this study, three wheat relatives, Psathyrostachys huashanica Keng, Dasypyrum villosum (L.), Leymus racemosus, Bin wheat, were used to co-transform receptor materials with a certain molar ratio to remove vector sequences and marker genes. (Leymus mollis) was used to clone the fructan-6-fructosyltransferase gene (6-SFT) from Neopsis huashanensis and L. villosa by genome walking and RT-PCR, and the 6-SFT gene from Leymus chinensis and L. littoralis and the fructan-1-fructosyltransferase gene (1-FFT), L. huashanensis were cloned by RT-PCR and RACE. The sucrose-1-fructosyltransferase gene (1-SST) of new wheat straw was constructed by using p1300-35SN as vector, and the plant expression vector p1300-35SN-FBEs was constructed. The linear expression cassettes of GUS and bar genes corresponding to plasmid pAHC25 and pEasy-Blunt-bar were digested by enzyme digestion and purified. The immature embryos of common wheat Kenong 199 were transformed with bar or pAHC20 in parallel with Gus masser 1:1,1:2,1:3 in order to determine the optimum molar ratio of screening gene and target gene. The main results obtained in this study are as follows: 1. A variety of fructan synthase genes were isolated from Triticum aestivum. Four 6-SFT genes, named Ph-6-SFT (1851 bp), Dv-6-SFT (1863 bp) and Lr-6-SFT (1863 bp), were cloned from Triticum aestivum, Triticum villosum, Leymus chinensis and Littoral. And Lm-6-SFT (1866 bp); cloned three 1-FFT genes from New Wheat Grass, Triticum villosum and Leymus chinensis, named Ph-1-FFT (1989 bp), Dv-1-FFT (1950 bp) and Lr-1-FFT (1989 bp); cloned one 1-SST gene from New Wheat Grass, named Ph-1-SST (2001 bp); completed the construction of several fructose transferase gene vectors: Ph-1-SST, Ph-SST, Ph-SST - 1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes were inserted into p1300-35SN vectors respectively to obtain plant expression vectors of seven genes; 3. Molecular identification of fructose synthase gene transformed tobacco and transgenic plants: The constructed plant expression vectors p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT/Ph-6-SFTS6-SFTSr-6/Lr 518 transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transformation and hygromycin screening. Genomic PCR and RT-PCR were used to identify transgenic tobacco plants with 6 vectors. A total of 277 transgenic positive plants with 64,53,55,24,38,43 were obtained from each vector. 4. Identification of stress resistance of transgenic plants: Phenotypic analysis showed that the expression of these genes could enhance the tolerance of transgenic tobacco to drought, cold and salt stress. The results showed that the positive plants of transgenic plants had higher fructose, lower soluble sugar and proline content than those of the control plants of transgenic plants under drought and cold stress, while the positive plants of transgenic plants of Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes had lower soluble sugar and proline content. The contents of fructan, soluble sugar and proline in transgenic plants treated with drought or cold stress were significantly higher than those in transgenic plants under normal conditions. The contents of MDA in transgenic plants were not significantly increased after drought and cold stress, but those in control plants were significantly higher than those under normal conditions. These results indicated that the expression of Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes could significantly enhance the accumulation of osmoregulant substances such as fructose in transgenic plants, thus making transgenic plants more resistant to oxidative damage induced by drought and cold stress. The results showed that 6-SFT gene was more effective than 1-FFT gene in improving crop resistance to stress. There was no significant difference among physiological indexes of the same gene in different species. 6. Establishment of the optimum molar ratio of the minimal expression cassette co-transformation Wheat Mediated by gene gun: the complete plasmid or expression cassette co-transformation Wheat Mediated by gene gun, a total of 3964 T0 generation wheat resistant plants were obtained after screening of the herbicide phosphinothricin (PPT), and 70 presumptive GU plants were identified by PCR. The transformation efficiency statistics showed that 11 transgenic plants with complete plasmids were obtained, the transformation rate was 0.70%; 30 transgenic plants with Gus + Bar expression cassette, the transformation rate was 0.16-1.03%; 29 transgenic plants with Gus + pAHC20, the transformation rate was 0.09-0.98%. The results of Southern blot analysis showed that GUS gene had been successfully integrated into wheat genome. The establishment of the optimum molar ratio of expression cassette co-transformed wheat could provide reference for the genetic transformation of fructan-related genes in common wheat. 7. The plant expression vector p1301-Ph-6-SFT/Dv-6-SFT was introduced by gene gun. The minimal expression cassette co-transformation method was used to transform common wheat Kenong 199. Resistance screening, GUS staining and PCR detection of GUS gene were carried out on T0 generation plants. The results showed that the expression vector had been successfully transfected into wheat genome, and one transgenic positive strain was confirmed.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1
，
本文编号：2178906
[Abstract]:Abiotic stress is an important environmental factor affecting the growth and development of crops and limiting their yield. How crops respond to abiotic stress is a worldwide research topic. Fructan accumulation is conducive to enhancing the resistance of plants to stress. It is of great theoretical and practical significance to identify the genes involved in fructan biosynthesis, identify their functions and study the improvement of wheat stress resistance breeding. In this study, three wheat relatives, Psathyrostachys huashanica Keng, Dasypyrum villosum (L.), Leymus racemosus, Bin wheat, were used to co-transform receptor materials with a certain molar ratio to remove vector sequences and marker genes. (Leymus mollis) was used to clone the fructan-6-fructosyltransferase gene (6-SFT) from Neopsis huashanensis and L. villosa by genome walking and RT-PCR, and the 6-SFT gene from Leymus chinensis and L. littoralis and the fructan-1-fructosyltransferase gene (1-FFT), L. huashanensis were cloned by RT-PCR and RACE. The sucrose-1-fructosyltransferase gene (1-SST) of new wheat straw was constructed by using p1300-35SN as vector, and the plant expression vector p1300-35SN-FBEs was constructed. The linear expression cassettes of GUS and bar genes corresponding to plasmid pAHC25 and pEasy-Blunt-bar were digested by enzyme digestion and purified. The immature embryos of common wheat Kenong 199 were transformed with bar or pAHC20 in parallel with Gus masser 1:1,1:2,1:3 in order to determine the optimum molar ratio of screening gene and target gene. The main results obtained in this study are as follows: 1. A variety of fructan synthase genes were isolated from Triticum aestivum. Four 6-SFT genes, named Ph-6-SFT (1851 bp), Dv-6-SFT (1863 bp) and Lr-6-SFT (1863 bp), were cloned from Triticum aestivum, Triticum villosum, Leymus chinensis and Littoral. And Lm-6-SFT (1866 bp); cloned three 1-FFT genes from New Wheat Grass, Triticum villosum and Leymus chinensis, named Ph-1-FFT (1989 bp), Dv-1-FFT (1950 bp) and Lr-1-FFT (1989 bp); cloned one 1-SST gene from New Wheat Grass, named Ph-1-SST (2001 bp); completed the construction of several fructose transferase gene vectors: Ph-1-SST, Ph-SST, Ph-SST - 1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes were inserted into p1300-35SN vectors respectively to obtain plant expression vectors of seven genes; 3. Molecular identification of fructose synthase gene transformed tobacco and transgenic plants: The constructed plant expression vectors p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT/Ph-6-SFTS6-SFTSr-6/Lr 518 transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transformation and hygromycin screening. Genomic PCR and RT-PCR were used to identify transgenic tobacco plants with 6 vectors. A total of 277 transgenic positive plants with 64,53,55,24,38,43 were obtained from each vector. 4. Identification of stress resistance of transgenic plants: Phenotypic analysis showed that the expression of these genes could enhance the tolerance of transgenic tobacco to drought, cold and salt stress. The results showed that the positive plants of transgenic plants had higher fructose, lower soluble sugar and proline content than those of the control plants of transgenic plants under drought and cold stress, while the positive plants of transgenic plants of Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes had lower soluble sugar and proline content. The contents of fructan, soluble sugar and proline in transgenic plants treated with drought or cold stress were significantly higher than those in transgenic plants under normal conditions. The contents of MDA in transgenic plants were not significantly increased after drought and cold stress, but those in control plants were significantly higher than those under normal conditions. These results indicated that the expression of Ph-1-FFT, Dv-1-FFT, Lr-1-FFT, Ph-6-SFT, Dv-6-SFT and Lr-6-SFT genes could significantly enhance the accumulation of osmoregulant substances such as fructose in transgenic plants, thus making transgenic plants more resistant to oxidative damage induced by drought and cold stress. The results showed that 6-SFT gene was more effective than 1-FFT gene in improving crop resistance to stress. There was no significant difference among physiological indexes of the same gene in different species. 6. Establishment of the optimum molar ratio of the minimal expression cassette co-transformation Wheat Mediated by gene gun: the complete plasmid or expression cassette co-transformation Wheat Mediated by gene gun, a total of 3964 T0 generation wheat resistant plants were obtained after screening of the herbicide phosphinothricin (PPT), and 70 presumptive GU plants were identified by PCR. The transformation efficiency statistics showed that 11 transgenic plants with complete plasmids were obtained, the transformation rate was 0.70%; 30 transgenic plants with Gus + Bar expression cassette, the transformation rate was 0.16-1.03%; 29 transgenic plants with Gus + pAHC20, the transformation rate was 0.09-0.98%. The results of Southern blot analysis showed that GUS gene had been successfully integrated into wheat genome. The establishment of the optimum molar ratio of expression cassette co-transformed wheat could provide reference for the genetic transformation of fructan-related genes in common wheat. 7. The plant expression vector p1301-Ph-6-SFT/Dv-6-SFT was introduced by gene gun. The minimal expression cassette co-transformation method was used to transform common wheat Kenong 199. Resistance screening, GUS staining and PCR detection of GUS gene were carried out on T0 generation plants. The results showed that the expression vector had been successfully transfected into wheat genome, and one transgenic positive strain was confirmed.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S512.1
，
本文编号：2178906
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