以gbss、ssⅢ和ssⅡ为靶标的RNAi载体构建及转基因马铃薯植株的获得
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摘要
马铃薯(Solanum tuberosum L)是淀粉生产的重要原料,其淀粉具有优良的特性,广泛应用于工业、食品及化工行业。但在大多数应用领域中,一般要求淀粉具有较低的糊化温度和较弱的凝沉(老化)性,因而淀粉的糊化和凝沉特性成为其应用的重要参考指标,而这两种性质主要与直链淀粉与支链淀粉比例、淀粉链长以及淀粉的结构有关。因此,培育具有较低糊化温度和优良凝沉特性的品种,对于提高马铃薯淀粉的应用范围和促进产业的发展具有重要的意义。
随着人们对淀粉生物合成途径相关酶生化代谢途径及分子机理研究的不断深入,使得应用基因工程技术调控淀粉合成过程中相关酶基因的表达,从而使得降低淀粉糊化温度和提高冻融稳定性成为可能。为此,本研究开展了以RNAi技术对马铃薯块茎内源gbss、ssⅢ和ssⅡ基因进行同时干扰,期望通过改变淀粉合成相关酶的活性,进而改变块茎中积累的淀粉分子组成,获得糊化温度低且冻融稳定性更好的加工型马铃薯品种。经过研究,已取得了如下结果:
(1)应用blast及分子生物学软件DNAstar对gbss、ssⅡ和ssⅢ基因的cDNA序列同源性进行分析比较,分别从gbss、ssⅢ和ssⅡ基因cDNA片段ORF中筛选出了1～261、2 164～2 407、161～441之间的序列片段作为靶标,采用一步PCR法将靶标基因片段进行融合,得到了大小为785 bp的gbss、ssⅡ和ssⅢ三个基因片段的融合基因。
(2)为便于构建RNAi载体,分别以LS-8质粒(含gbss基因5'侧翼序列)、LS-4质粒(含patatin启动子序列)为模板,亚克隆了马铃薯块茎颗粒结合型淀粉合成酶gbss基因5'侧翼序列及马铃薯patatin启动子,并在这两个启动子5'和3'加入了Sac I和Xho I限制性酶切位点。
(3)分别构建了以gbss基因5'侧翼序列和patatin组织特异性启动子驱动的含有“正向gbs3s2融合片段-pdk内含子-反向gbs3s2融合片段”的植物表达载体pART-GF、pART-PF,并将其成功导入根癌农杆菌LBA4404。
(4)选用马铃薯“NHD3”、“NF”和“NC”的茎段作为外植体材料,在MS培养基基本成分的基础上,对生长激素的浓度和配比进行了优化,筛选出有利于受体材料高效再生的培养基配方,其中有利于茎段愈伤诱导的培养基为:MS0 + 1.0 mg/L 6-BA + 0.3 mg/L NAA(NHD3);MS0 + 1.0 mg/L 6-BA + 0.2 mg/L NAA(NC);MS0 + 1.0 mg/L 6-BA + 0.4 mg/L NAA(NF)。初步建立并优化了遗传转化体系。
(5)用携带pART-GF植物表达载体的农杆菌对NHD3、NF和NC三个优良马铃薯品种进行了遗传转化,初步获得了抗性植株,经PCR检测有10株呈阳性,其中在NC、NF和NHD3品种上分别得到的转基因阳性植株为2株、3株和5株。
Potato(Solanum tuberosum L) is an important raw material for starch production.So potato starch has superior trait and have been an important raw material for many industrial applications,such as industry,food service industry and chemical engineering.In the most application domains,the potato starch is requested to possess lower-gelatinization temperature and faintish retrogradation,so they have been the important index.However,the two natures are related to the chain length,the ratio of amylose content to amylopectin and the structure of starch.Therefore,to cultivate a kind of potato with the low gelatinization temperature and faintish retrogradation has important significance for improving the application range of potato starch and the promote the industrial development.In the past few years,with the further reserch of enzemy related to the biosynthesis and molecular mechanism of potato starch,it will be possible to use technique of genetic engineering to regulation the key enzymatic gene expression for improving the content,branching degree and the length of amylopectin.For the above mentioned goals,we do some research and expect to get the new variety of potato which has lower-gelatinization temperature and faintish retrogradation nature by silencing the gbss,ssⅢand ssⅡendogenous genes of potato tuber with RNA interference.The following are the main works we did:
(1)The gbss,ssⅢand ssⅡgene sequence homology of potato were analysed with the blast method and molecular biology software DNA Star,the gbss(1～261),ssⅢ(2 164～2 407) and ssⅡ(161～441)fragments were selected from these genes and were taken as target.At the same time,the three acquainted gene fragments were fused by the one-step PCR method,the fused gene we obtained is a 785 bp fragment.
(2)In order to construction of RNAi expression vector,the 5' flanking region of gbss gene was sub-cloned from plasmid LS-8 and the tuber-specific-promoter patatin was also sub-cloned from plasmid LS-4,the two kinds of promoter were inserted the restriction site Sac I at the 5' end and the restriction site Xho I at the 3' end.
(3)Construction of ihRNAi Expression Vectors of“forward direction gbs3s2-pdk(intro)-reverse gbs3s2”regulating by the 5' flanking region of gbss gene and patatin tissue specificity promotor for breeding a low gelatinization temperature potato.
(4)In this study,the item of potato as the explant,Some parameters of Agrobaterium tumefaciens mediated inheritable transfromation was established.Based on the MS medium,the density and the assignment of the hormones was optimized,finally to confirm the optimum medium in favor of the growth of the callus:MS0+1.0 mg/L 6-BA +0.3 mg/L NAA(NHD3);MS0+1.0 mg/L 6-BA +0.2 mg/L NAA (NC);MS0 + 1.0 mg/L 6-BA +0.4 mg/L NAA(NF).
(5)The pARTGF was transferred to three fine potato varieties plants by agrobacterium tumefaciens system.the resistance plants were obtained and the results of pcr indicated that 10 regenerated plants were positive,there were 2 NC plants,3 NF plants and 5 NHD3 plants among the total.
随着人们对淀粉生物合成途径相关酶生化代谢途径及分子机理研究的不断深入,使得应用基因工程技术调控淀粉合成过程中相关酶基因的表达,从而使得降低淀粉糊化温度和提高冻融稳定性成为可能。为此,本研究开展了以RNAi技术对马铃薯块茎内源gbss、ssⅢ和ssⅡ基因进行同时干扰,期望通过改变淀粉合成相关酶的活性,进而改变块茎中积累的淀粉分子组成,获得糊化温度低且冻融稳定性更好的加工型马铃薯品种。经过研究,已取得了如下结果:
(1)应用blast及分子生物学软件DNAstar对gbss、ssⅡ和ssⅢ基因的cDNA序列同源性进行分析比较,分别从gbss、ssⅢ和ssⅡ基因cDNA片段ORF中筛选出了1～261、2 164～2 407、161～441之间的序列片段作为靶标,采用一步PCR法将靶标基因片段进行融合,得到了大小为785 bp的gbss、ssⅡ和ssⅢ三个基因片段的融合基因。
(2)为便于构建RNAi载体,分别以LS-8质粒(含gbss基因5'侧翼序列)、LS-4质粒(含patatin启动子序列)为模板,亚克隆了马铃薯块茎颗粒结合型淀粉合成酶gbss基因5'侧翼序列及马铃薯patatin启动子,并在这两个启动子5'和3'加入了Sac I和Xho I限制性酶切位点。
(3)分别构建了以gbss基因5'侧翼序列和patatin组织特异性启动子驱动的含有“正向gbs3s2融合片段-pdk内含子-反向gbs3s2融合片段”的植物表达载体pART-GF、pART-PF,并将其成功导入根癌农杆菌LBA4404。
(4)选用马铃薯“NHD3”、“NF”和“NC”的茎段作为外植体材料,在MS培养基基本成分的基础上,对生长激素的浓度和配比进行了优化,筛选出有利于受体材料高效再生的培养基配方,其中有利于茎段愈伤诱导的培养基为:MS0 + 1.0 mg/L 6-BA + 0.3 mg/L NAA(NHD3);MS0 + 1.0 mg/L 6-BA + 0.2 mg/L NAA(NC);MS0 + 1.0 mg/L 6-BA + 0.4 mg/L NAA(NF)。初步建立并优化了遗传转化体系。
(5)用携带pART-GF植物表达载体的农杆菌对NHD3、NF和NC三个优良马铃薯品种进行了遗传转化,初步获得了抗性植株,经PCR检测有10株呈阳性,其中在NC、NF和NHD3品种上分别得到的转基因阳性植株为2株、3株和5株。
Potato(Solanum tuberosum L) is an important raw material for starch production.So potato starch has superior trait and have been an important raw material for many industrial applications,such as industry,food service industry and chemical engineering.In the most application domains,the potato starch is requested to possess lower-gelatinization temperature and faintish retrogradation,so they have been the important index.However,the two natures are related to the chain length,the ratio of amylose content to amylopectin and the structure of starch.Therefore,to cultivate a kind of potato with the low gelatinization temperature and faintish retrogradation has important significance for improving the application range of potato starch and the promote the industrial development.In the past few years,with the further reserch of enzemy related to the biosynthesis and molecular mechanism of potato starch,it will be possible to use technique of genetic engineering to regulation the key enzymatic gene expression for improving the content,branching degree and the length of amylopectin.For the above mentioned goals,we do some research and expect to get the new variety of potato which has lower-gelatinization temperature and faintish retrogradation nature by silencing the gbss,ssⅢand ssⅡendogenous genes of potato tuber with RNA interference.The following are the main works we did:
(1)The gbss,ssⅢand ssⅡgene sequence homology of potato were analysed with the blast method and molecular biology software DNA Star,the gbss(1～261),ssⅢ(2 164～2 407) and ssⅡ(161～441)fragments were selected from these genes and were taken as target.At the same time,the three acquainted gene fragments were fused by the one-step PCR method,the fused gene we obtained is a 785 bp fragment.
(2)In order to construction of RNAi expression vector,the 5' flanking region of gbss gene was sub-cloned from plasmid LS-8 and the tuber-specific-promoter patatin was also sub-cloned from plasmid LS-4,the two kinds of promoter were inserted the restriction site Sac I at the 5' end and the restriction site Xho I at the 3' end.
(3)Construction of ihRNAi Expression Vectors of“forward direction gbs3s2-pdk(intro)-reverse gbs3s2”regulating by the 5' flanking region of gbss gene and patatin tissue specificity promotor for breeding a low gelatinization temperature potato.
(4)In this study,the item of potato as the explant,Some parameters of Agrobaterium tumefaciens mediated inheritable transfromation was established.Based on the MS medium,the density and the assignment of the hormones was optimized,finally to confirm the optimum medium in favor of the growth of the callus:MS0+1.0 mg/L 6-BA +0.3 mg/L NAA(NHD3);MS0+1.0 mg/L 6-BA +0.2 mg/L NAA (NC);MS0 + 1.0 mg/L 6-BA +0.4 mg/L NAA(NF).
(5)The pARTGF was transferred to three fine potato varieties plants by agrobacterium tumefaciens system.the resistance plants were obtained and the results of pcr indicated that 10 regenerated plants were positive,there were 2 NC plants,3 NF plants and 5 NHD3 plants among the total.
引文
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