沉默马铃薯Sgt基因家族对块茎GAs积累的影响

发布时间：2018-07-06 07:08

本文选题：马铃薯 + 糖苷生物碱　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：糖苷生物碱(GAs)具有抑制病原微生物侵袭、拒避昆虫采食、抗渗透胁迫和化感效应,但马铃薯(Solanum tuberosum)块茎中过量积累会影响到食用风味和安全性。因此,通过分子调控手段特异性地降低块茎中GAs积累,对于提高马铃薯抗逆性并满足食用安全性至关重要。GAs合成途径的末端酶为茄啶-糖基转移酶(Solanidine glycosyltransferase,Sgt)家族的三个成员(Sgt1、Sgt2和Sgt3),它们的表达水平和活性影响到GAs的合成速率和水平。目前,对于马铃薯块茎GAs合成代谢调控,仅见到对于Sgt家族成员其中之一的抑制研究,但是对Sgt家族成员的单基因实施调控,很难有效降低块茎中GAs的积累。为此,本研究采用RNAi技术,以GAs合成代谢途径三个末端酶基因为靶标,构建块茎特异性启动子Patatin驱动的RNAi载体,采用农杆菌介导法转化2个马铃薯基因型,用以鉴定特异抑制三个Sgt基因家族对于块茎中GAs积累的影响,为采用分子调控技术培育块茎GAs含量低的马铃薯种质提供理论依据和参考。目前研究取得了如下结果:1.采用生物信息学方法,对GAs合成途径3个末端酶基因进行寻靶,找到了敏感靶标区段,采用重叠PCR技术将3个基因敏感片段融合,得到正向片段F和反向片段R。然后将二者分别插入载体pHANNIBAL内含子(intron)两侧,获得重组质粒pHAI-FR,再使用Patatin启动子替换pHANNIBAL所携带的CaMV 35s启动子;最后将“Patatin P-正向融合片段-pdk-反向融合片段-OCS-ter”整体酶切后插入载体pCEPSPS,最终得到含融合基因ABC反向重复序列的植物表达载体pCEI-PFR,并利用冻融转化法将所获得的植物表达载体整合到农杆菌LBA4404中;2.采用农杆菌介导法转化2个马铃薯基因型(庄薯3号和费乌瑞它),使用农杆菌菌液对其无腋芽茎段进行侵染,经过草甘膦筛选和PCR检测,得到了10株(庄薯3号)和11株(费乌瑞它)转融合基因反向重复片段表达呈阳性的植株,阳性植株率为0.50%-0.90%(庄薯3号)和0.75%-0.90%(费乌瑞它);3.使用RT-qPCR技术测定转化植株内各基因的表达进行检测,结果证实,转化植株块茎内三个Sgt基因的表达量分别降低了32%-60%(Sgt1)、29%-55%(Sgt2)和25%-66%(Sgt3),而草甘膦基因的表达量增加了48%-135%;但叶片中Sgts基因的表达量未有显著变化;4.利用分光光度法对马铃薯GAs含量进行了测定。与野生马铃薯相对比,转基因植株地下部分的GAs含量降低了41%-59%(庄薯3号)和36%-62%(费乌瑞它);而地上部分的GAs含量并无显著减少。
[Abstract]:Glycoside alkaloids (gas) can inhibit the invasion of pathogenic microorganisms, avoid insect feeding, resist osmotic stress and allelopathy, but excessive accumulation of potato (Solanum tuberosum) tubers will affect the food flavor and safety. Therefore, the accumulation of gas in tubers was specifically reduced by means of molecular regulation. The terminal enzyme of Solanidine glycosyltransferase (Solanidine glycosyltransferase Sgt) family (Sgt1, Sgt2 and Sgt3) is the terminal enzyme of potato biosynthesis pathway. The expression and activity of these enzymes affect the synthesis rate and level of gas. At present, only one of the Sgt family members is inhibited by the regulation of the synthesis and metabolism of potato tubers, but it is difficult to effectively reduce the accumulation of gas in the tubers by regulating the single gene of the Sgt family members. In this study, RNAi was used to construct tuber specific promoter Patatin driven RNAi vector, and two potato genotypes were transformed by Agrobacterium tumefaciens mediated transformation. It was used to identify the effects of specific inhibition of three Sgt gene families on the accumulation of gas in tubers, and to provide theoretical basis and reference for the cultivation of potato germplasm with low gas content in tubers by molecular control techniques. The results of the present study are as follows: 1. The three terminal enzyme genes of gas synthesis pathway were targeted by bioinformatics, and the sensitive target region was found. The positive fragment F and the reverse fragment R3 were fused by overlapping PCR technique. Then the recombinant plasmid pHAI-FRwas obtained by inserting them into the (intron) of the intron of pH ANNIBAL, and the CaMV 35s promoter carried by pH ANNIBAL was replaced by the patatin promoter. Finally, "Patatin P- forward fusion fragment-pdk- reverse fusion fragment-OCS-ter" was digested and inserted into the vector pCEPSPS.The plant expression vector pCEI-PFRcontaining ABC reverse repeat sequence of fusion gene was obtained, and the plant was obtained by freeze-thaw transformation. The expression vector was integrated into Agrobacterium tumefaciens LBA4404. Two potato genotypes (Zhuangshu 3 and Fiurita) were transformed by Agrobacterium tumefaciens (Agrobacterium tumefaciens) and infected with Agrobacterium tumefaciens. Ten (Zhuangshu 3) and 11 (Fiurita) transgenic plants with positive reverse repeat expression of fusion gene were obtained. The positive rates were 0.50-0.90% (Zhuangshu 3) and 0.75 -0.90% (Fiurita). RT-qPCR technique was used to detect the expression of the genes in the transformed plants. The expression of three SGT genes in tubers of transformed plants decreased by 32-60% (SgT1) 29-55% (Sgt2) and 25-66% (Sgt3), while glyphosate gene expression increased by 48-135%, but there was no significant change in Sgts gene expression in leaves. The content of gas in potato was determined by spectrophotometry. Compared with wild potato, gas content in underground parts of transgenic plants was decreased by 41-59% (Zhuangshu No. 3) and 36-62% (Fiurita), but no significant decrease was found in aboveground parts.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S532

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