【作者】 朱青；

【导师】 柳俊；

【作者基本信息】 华中农业大学 ， 遗传学， 2008， 博士

【摘要】 马铃薯是世界第四大粮食作物,其块茎除用作鲜食外,还作为工业原料加工各种食品和其它淀粉产品,其中薯片和薯条等加工产品的生产在马铃薯加工业中占据重要位置。为了延长加工周期,低温贮藏是最经济有效的方法,然而低温导致还原糖的大量累积,使得在高温油炸时还原糖与自由氨基酸发生褐化反应,严重影响了加工产品的品质。马铃薯油炸加工品质改良的基因工程目前已作为重要的育种途径在国内外展开研究,为使目的基因在不干扰马铃薯正常生长而只在贮藏期间低温诱导表达,低温诱导的块茎特异表达启动子成为油炸加工品质基因工程育种的迫切需要。本研究在克隆相关低温诱导表达基因启动子的基础上,构建具有低温诱导活性的块茎特异表达融合启动子,评价其在马铃薯基因工程中潜在的应用价值。并在此基础上,对马铃薯中调控低温诱导启动子活性的蛋白质因子进行了初步研究,取得的主要研究结果如下:1.低温启动子诱导活性分析:采用PCR方法分别从拟南芥和马铃薯中克隆了低温诱导cor15α基因的启动子和ci21A基因的启动子。序列比较分析显示,cor15α基因的启动子含有目前研究确定的低温响应元件“LTRE”,而ci21A基因的启动子中没有该元件。将cor15α基因启动子和ci21A基因启动子分别与GUS基因连接,构建了表达载体pLB和pCI21A,采用根癌农杆菌介导的转化技术转化烟草并获得转基因植株。转基因烟草的GUS染色和GUS活性荧光定量分析结果显示,cor15α基因启动子和ci21A基因启动子在烟草中都具有表达功能,总体表达强度分析显示,ci21A基因启动子表达强度高于cor15α基因启动子,但cor15α基因启动子对低温诱导的敏感性强于ci21A基因启动子。2.cor15α基因启动子在马铃薯中表达活性鉴定:为了检测cor15α基因启动子在马铃薯中是否具有低温诱导活性,本研究将表达载体pLB通过农杆菌介导转化马铃薯“鄂马铃薯3号(E3)”并获得转化再生植株。转基因马铃薯的GUS染色和GUS活性荧光定量分析结果证明,cor15α基因启动子在马铃薯中仍具有低温诱导表达特性。在没有低温处理的对照组中,各被测样品组织中均检测不到GUS的酶活性,低温处理后,在茎,叶,块茎和匍匐茎组织中均可以检测到不同强度的GUS活性,其中叶片和块茎中的活性相对较高。3.cor15α基因启动子和块茎特异启动子(CIPP)的特异核心启动子区域表达活性鉴定:实验将cor15α基因启动子的-297/+70(含有一个低温响应元件LTRE)与GUS基因连接,构建了转化载体pL297-121,将CIPP启动子的-340/+19(含有块茎特异表达调控序列TSSR)与GUS基因连接,构建了转化载体pC340-121。pL297-121和pC340-121分别转化马铃薯E3获得转基因植株,分析显示,载体pL297-121在除根以外所有被检测的组织中均具有低温诱导表达功能,但表达强度低于完整cor15α基因启动子。pC340-121在块茎中的表达活性明显高于茎,根和匍匐茎,而在叶片中则始终未检测到它的活性。同时低温处理后,pC340-121的活性在所有被测组织中均没有变化,表明该TSSR序列的调控活性不受低温环境的影响。4.低温诱导的马铃薯块茎特异启动子构建与表达功能鉴定:以低温诱导cor15α基因的启动子和马铃薯块茎特异启动子CIPP为基础,采用重叠延伸PCR的方法,将cor15α基因启动子中含有低温响应元件LTRE的-297/-42和-297/+70片段分别与CIPP启动子中含有块茎特异表达序列TSSR的-340/+19和-340/-28片段进行融合,按照核心序列相对于TATA-box位置的不同,构建了2个融合启动子pCL(TSSR靠近TATA-box)和pLC(LTRE靠近TATA-box),并将2个融合启动子分别与GUS基因连接,通过农杆菌介导的基因转化,将融合启动子pCL和pLC导入E3获得转基因植株。分析结果显示,融合启动子pCL和pLC均具有表达功能,但核心功能序列相对于TATA-box的位置不同其表达强度具有显著差异。块茎特异表达序列TSSR靠近TATA-box的pCL,GUS表达强度显著高于低温诱导核心启动子序列LTRE靠近TATA-box的pLC,特别是在块茎和匍匐茎中表现更为突出。5.马铃薯St-CBF转录因子的鉴定:本研究根据已报道的EST序列设计引物,采用基于PCR的技术从马铃薯两个基因型(E3和CW2-1)中克隆到St-CBF基因的蛋白质编码区序列。序列分析显示该片段长600bp,编码199个氨基酸,具有CBF/DREB转录因子的保守结构域ERF/AP2,同时还具有一个富含丝氨酸/苏氨酸的保守区域,一个核定位信号NLS(PKRPAGRKKFRETRHP)和一个保守的DSAW-Motif。该St-CBF具备CBF1/DREB1类转录因子的典型特征。氨基酸序列比对分析表明,St-CBF与许多植物中的CBF/DREB转录因子蛋白具有较高的同源性。将St-CBF基因克隆到pGEX-6P-1上与谷胱苷肽-S-转移酶进行融合,并在大肠杆菌中BL21(DE3)中成功进行了融合蛋白的表达。凝胶阻滞实验表明,该融合蛋白能够与cor15α基因启动子中的LTRE序列发生特异地结合,初步证明马铃薯St-CBF蛋白具有CBF/DREB类转录因子的功能。Southern杂交显示St-CBF基因在马铃薯基因组中只有一个位点。RT-PCR结果显示St-CBF基因在低温处理15min即开始在叶片中表达,而且在整个低温处理期间(12h)持续表达,表明马铃薯St-CBF基因可能参与调节低温诱导基因的表达。更多还原

【Abstract】 The potato (Solarium tuberosum L.) is the fourth food crop in the world. In addition to its table use, potato tubers are important industrial materials for producing food and starch product. The production of chips and fries have taken an important position in potato precessing industry. To extend its processing period, storing tubers at low temperature is the most efficient and economic way. However, in cold-stored tubers, the accumulation of reducing sugar caused by low temperature results in an unacceptable dark and bitter product because of the browning reaction between reducing sugars and free amino acid groups at high frying temperatures. Now, gene engineering approaches have been important breeding strategies for the improvement of processing quality of potato products (fries and chips) worldwide. However, these approaches require gene expression in tubers at low temperature in order to avoid potential pleiotropic effects on the growth and development of potato plants. To achieve this gene expression manner, promoters with specificity to tubers as well as inducible activity under low temperature are definitely required in potato gene engineering to improve the processing quality of potato products.Based on the cloning of cold-induced promoters, present research constructed synthetic promoters with tuber-specific and cold-responsive patterns, investigated their performance in transgenic potato and their potential applications in potato gene engineering. Moreover, present research performed pilot study to explore putative transcription factors regulating the activities of cold-induced promoters in potato. The main results achieved are as following:1. The cold-inducibility analysis of cold-inducible promoters: By PCR, the cold-inducible promoters of cor15a gene and ci21A gene were amplified from Arabidopsis thaliana (cv. Columbia) genomic DNA and that of Solarium tuberosum (cv. E3) respectively. Promoter sequences analysis showed that cor15a promoter contained the identified low temperature responsive element "LTRE", while ci21A gene promoter did not. Expression vectors, pLB and pCI21A, were constructed by inserting the cor15a promoter and the ci21A promoter into the upstream of GUS gene followed by NOS terminator in pBI121 respectively. Then the pLB and pCI21A were transformed separately into tobacco via Agrobacterium mediated transformation. Transgenic plants were confirmed by PCR and Southern blot. Histochemical staining and fluorescent quantitative analysis of GUS activity in the tested transgenic plants indicated that both the two promoters could be functional in tobacco. The promoter strength and cold-inducibility analysis showed that the expression strength of ci21A promoter was higher than that of corl5a promoter, but the cold-inducibility of cor15a promoter was higher than that of ci21A promoter.2. The functional identification of cor15a promoter in potato: to investigate whether cor15a promoter could be cold-induced in potato, the construct of pLB was introduced into potato "E3" genome via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were analyzed by PCR and Southern blot. GUS histochemical staining and GUS fluorescent quantitative analysis of the tested transgenic potato plants proved that cor15a promoter could maintain its cold-inducibility in potato. Without cold treatment, no GUS activity could be detected in all of the potato tissues. After cold treatment, various GUS activities were detected in stems, leaves, tubers and stolons, moreover the GUS activities in leaves and tubers were relatively high.3. The functional investigation of the LTRE from cor15a promoter and the TSSR from tuber-specific promoter CIPP: The -297/+70 fragment of cor15a promoter containging LTRE (low temperature responsive element) and the -340/+19 fragment of CIPP containing TSSR (tuber-specific and sucrose responsive sequence) were cloned into the upstream of GUS in pBI121 to generate transformation constructs pL297-121 and pC340-121 respectively, which were subsequently introduced into potato "E3" genome via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were analyzed by PCR and Southern blot. GUS fluorescent quantitative analysis in transgenic potatoes showed that pL297-121 had cold inducibility in all the tested potato tissues except roots, but its promoter strength was less than that of the full length cor15a promoter. pC340-121 showed higher promoter activity in tubers than in stems, roots and stolons, and no activity was detected in leaves. After cold treatment, the promoter activity of pC340-121 did not show obvious change, indicating the function of TSSR was not affected by low temperature.4. Construction and functional identification of cold-induced and tuber-specific synthetic promoters in potato: By overlapping extension PCR, two chimeric promoters, pCL and pLC, were constructed with different combinations of cor15a promoter fragments, -297/-42 and -297/+70, containing the LTRE (low temperature responsive element) and CIPP promoter fragments, -340/+19 and -340/-28, containing the TSSR (tuber-specific and sucrose-responsive sequence).The TSSR in pCL is closer to the TATA-box than in pLC, in which the LTRE is close to the TATA-box. The two synthetic promoters were cloned into the upstream of GUS gene in pBI121 respectively and then introducede into potao "E3" via Agrobacterium mediated transfermation of microtubers. The transgenic potato plants were obtained by PCR and Southern blot. The cold-inducible and tuber-specific activities of synthetic promoters were investigated by quantitative analysis of GUS activity in transgenic potatoes and the results showed that both the two synthetic promoters could be functional in potato. But pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20℃) or low temperature (2℃), especially in tubers and stolons. This result suggested that the promoter activity was closely associated with the position of the two functional cis-elements, TSSR and LTRE, to the TATA-box.5. The indentification of St-CBF thanscription factor in potato: An encoding sequence of St-CBF gene was amplified from leaves of potato (E3 and CW2-1) by RT-PCR, using primers designed according to the released EST sequence (accession number CK860811). The DNA sequence of St-CBF is of 600bp in length, encoding a protein of 199 amino acids. The analysis of deduced amino acids sequences of St-CBF showed it was high homologous to other CBF/DREB proteins and contained a typical domain ERF/AP2 of CBF/DREB-type proteins. Moreover, three conserved sequences were also found: the conserved Ser/Thr-rich region located at the N-end, the conserved CBF1/DREB1-type NLS and the conserved DSAW-motif surrounding the ERF/AP2 domain. Tthe St-CBF gene was classed into the CBF1/DREB1 category for the presence of conserved NLS and DSAW-motif, which distinguishing CBF1/DREB1-type proteins from other ERF/AP2 proteins. The encoding sequence of St-CBF was cloned into the expression vector of pGEX-6P-1 and expressed successfully as GST (Glutathione S-Transferase) fusion protein in E. coli. The GST-St-CBF fusion protein could bind specifically to the LTRE sequence from cor15a promoter in vitro, indicating that the St-CBF protein could act as CBF/DREB transcription factors. The Southern blot results showed the St-CBF gene had only one allele in potato genome. In leaves, the expression of St-CBF gene was detected within 15min after potato plants exposed to low temperature (2℃) and remained detectable during low temperature treatment (up to 12h). This result indicted that the St-CBF gene was involved in the gene expression of potato in response to the low temperature stress.更多还原