小麥近緣屬植物果聚糖合成相關(guān)基因的克隆和功能驗證及小麥共轉(zhuǎn)化體系的優(yōu)化
發(fā)布時間：2018-08-12 11:23
【摘要】：非生物脅迫是影響作物生長發(fā)育、限制其產(chǎn)量的重要環(huán)境因子,作物如何應(yīng)對非生物脅迫是世界性的研究課題。果聚糖的積累有利于增強植物對逆境脅迫的抵抗能力。小麥近緣屬植物具有抗旱、抗寒、抗鹽及耐瘠薄等優(yōu)良特性。因此,克隆其果聚糖合成相關(guān)基因,鑒定其功能并進(jìn)行小麥抗逆育種改良研究具有重要的理論和現(xiàn)實意義。轉(zhuǎn)基因植物的載體骨架序列及抗性標(biāo)記是轉(zhuǎn)基因生物安全性爭論的焦點,是限制轉(zhuǎn)基因植物商業(yè)化的重要因素�；驑尳閷�(dǎo)的最小表達(dá)盒(目的基因和篩選基因)按一定摩爾比共轉(zhuǎn)化受體材料可能是有效去除載體序列和標(biāo)記基因的一種高效的遺傳轉(zhuǎn)化法。本研究以小麥近緣屬植物華山新麥草(Psathyrostachys huashanica Keng)、簇毛麥(Dasypyrum villosum(L.)),大賴草(Leymus racemosus)、濱麥(Leymus mollis)為材料,利用genome walking結(jié)合RT-PCR技術(shù)克隆到華山新麥草和簇毛麥果聚糖-6-果糖基轉(zhuǎn)移酶基因(6-SFT);利用RT-PCR結(jié)合RACE技術(shù)克隆到大賴草和濱麥6-SFT基因及華山新麥草、簇毛麥、大賴草果聚糖-1-果糖基轉(zhuǎn)移酶基因(1-FFT),華山新麥草蔗糖-1-果糖基轉(zhuǎn)移酶基因(1-SST),并對其進(jìn)行基因結(jié)構(gòu)和序列分析;以p1300-35SN為載體,構(gòu)建植物表達(dá)載體p1300-35SN-FBEs。通過農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草,對轉(zhuǎn)基因植株進(jìn)行抗旱、抗寒和抗鹽性鑒定,并測定與果聚糖含量相關(guān)的重要生理指標(biāo)。以質(zhì)粒pAHC25為參照,將質(zhì)粒pAHC25和pEasy-Blunt-bar所對應(yīng)的gus和bar基因的線性表達(dá)盒通過酶切消化并純化回收,將bar或pAHC20與gus按摩爾比1:1、1:2、1:3平行地共轉(zhuǎn)化普通小麥科農(nóng)199幼胚,以期確定篩選基因與目標(biāo)基因的最佳摩爾比,建立安全高效的基因槍介導(dǎo)的普通小麥表達(dá)盒共轉(zhuǎn)化體系。本文得到的主要研究結(jié)果如下:1.分離到多種果聚糖合成酶基因:克隆到來自華山新麥草、簇毛麥、大賴草和濱麥的4個6-SFT基因,分別命名為Ph-6-SFT(1851 bp)、Dv-6-SFT(1863 bp)、Lr-6-SFT(1863 bp)和Lm-6-SFT(1866 bp);克隆到華山新麥草、簇毛麥、大賴草的3個1-FFT基因,分別命名為Ph-1-FFT(1989 bp)、Dv-1-FFT(1950 bp)和Lr-1-FFT(1989 bp);克隆到華山新麥草的1個1-SST基因,命名為Ph-1-SST(2001 bp);2.完成多個果聚糖果糖基轉(zhuǎn)移酶基因載體構(gòu)建:將Ph-1-SST、Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因分別插入到p1300-35SN載體獲得7個基因的植物表達(dá)載體;3.果聚糖合成酶基因轉(zhuǎn)化煙草及轉(zhuǎn)基因植株的分子鑒定:將所構(gòu)建的植物表達(dá)載體p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT/Ph-6-SFT/Dv-6-SFT/Lr-6-SFT通過農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草,經(jīng)潮霉素篩選后每個載體分別獲得113、98、101、65、69、72共518株轉(zhuǎn)基因煙草植株,應(yīng)用基因特異性引物對6個載體的轉(zhuǎn)基因煙草植株進(jìn)行基因組PCR、RT-PCR鑒定,每個載體分別獲得64、53、55、24、38、43共277株轉(zhuǎn)基因陽性植株。4.轉(zhuǎn)基因植株抗逆性鑒定:表型分析結(jié)果表明,這些基因的表達(dá)都能夠增強轉(zhuǎn)基因煙草對旱、冷、鹽脅迫的耐受能力。不同物種的6-SFT基因比1-FFT基因抗逆效果好,不同物種的同一基因抗逆性無明顯差異。相比之下,Ph-6-SFT和Dv-6-SFT基因的抗逆效果較好。5.轉(zhuǎn)基因植株生理指標(biāo)的測定:生理指標(biāo)測定結(jié)果表明,在旱及冷脅迫處理條件下,與轉(zhuǎn)空載體的對照植株相比轉(zhuǎn)Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因的陽性植株具有較高的果聚糖、可溶性糖及脯氨酸含量;較低的丙二醛(Malondialdehyde,MDA)含量。旱或冷脅迫處理后的轉(zhuǎn)基因植株果聚糖、可溶性糖和脯氨酸含量顯著高于正常條件下轉(zhuǎn)基因植株的果聚糖、可溶性糖和脯氨酸含量。旱和冷脅迫后轉(zhuǎn)基因植株丙二醛含量增加不顯著,而對照植株丙二醛含量顯著增加。這些結(jié)果表明Ph-1-FFT、Dv-1-FFT、Lr-1-FFT、Ph-6-SFT、Dv-6-SFT和Lr-6-SFT基因的表達(dá)均能顯著增強轉(zhuǎn)基因植株果聚糖等滲透調(diào)節(jié)物質(zhì)的積累從而使轉(zhuǎn)基因植株能更好的抵抗旱和冷脅迫所致的氧化損傷。轉(zhuǎn)不同基因的轉(zhuǎn)基因植株各生理指標(biāo)的比較結(jié)果表明,與1-FFT基因相比6-SFT基因?qū)μ岣咦魑锬婢趁{迫抗性更有效。不同物種的同一基因各生理指標(biāo)之間的差異不顯著。相比之下,Dv-6-SFT和Ph-6-SFT基因?qū)μ岣咧参锏目购�、抗寒效果較好。以上試驗結(jié)果表明Dv-6-SFT和Ph-6-SFT基因可應(yīng)用到作物抗逆育種中。6.基因槍介導(dǎo)的最小表達(dá)盒共轉(zhuǎn)化小麥最佳摩爾比的確立:基因槍介導(dǎo)的完整質(zhì)�；虮磉_(dá)盒共轉(zhuǎn)化普通小麥,除草劑草丁膦(phosphinothricin,PPT)篩選后共獲得3964株T0代小麥抗性植株,PCR鑒定共獲得70株推定的GUS陽性植株,GUS染色檢測共獲得56株陽性植株。轉(zhuǎn)化效率統(tǒng)計結(jié)果顯示,獲得完整質(zhì)粒的轉(zhuǎn)基因植株11株,轉(zhuǎn)化率為0.70%;gus+bar表達(dá)盒的轉(zhuǎn)基因植株30株,轉(zhuǎn)化率為0.16-1.03%;gus+pAHC20的轉(zhuǎn)基因植株29株,轉(zhuǎn)化率為0.09-0.98%。其中pAHC20/bar:gus摩爾比為1:2時轉(zhuǎn)化率顯著高于其他組合。Southern blot分析結(jié)果表明,gus基因已成功整合到小麥基因組中。表達(dá)盒共轉(zhuǎn)化小麥最佳摩爾比的確立為普通小麥遺傳轉(zhuǎn)化果聚糖相關(guān)基因提供參考依據(jù)。7.將植物表達(dá)載體p1301-Ph-6-SFT/Dv-6-SFT通過基因槍介導(dǎo)的最小表達(dá)盒共轉(zhuǎn)化法轉(zhuǎn)化普通小麥科農(nóng)199,對T0代植株進(jìn)行抗性篩選、葉片GUS染色、gus基因的PCR檢測,結(jié)果表明已成功將表達(dá)載體轉(zhuǎn)入小麥基因組,并且確認(rèn)各獲得1株轉(zhuǎn)基因陽性株系。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S512.1
，
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S512.1
，
本文編號：2178906
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