【作者】 李月；

【导师】 孙杰； 谢宗铭；

【作者基本信息】 石河子大学 ， 作物遗传育种， 2013， 博士

【摘要】 1.研究目的植物在生长发育过程中面临的各种不利的环境胁迫，包括生物胁迫和非生物胁迫（高盐、干旱、低温等），都对植物的生长发育和生产力造成影响。非生物胁迫是导致农作物减产的主要原因，农作物平均产量损失超过50%。在作物基因工程改良中，增加一个关键的转录因子的调控能力，能同时激活多个抗逆功能基因表达，从而能够提高作物综合抗逆性。Trihelix转录因子是植物特有的转录因子，在植物逆境信号传递过程中发挥着关键作用，能与其顺式作用元件结合，启动抗逆相关基因的表达，增强植物的逆境耐受性。棉花既是主要的纤维作物，也是重要的油料作物，干旱、盐碱、低温等非生物逆境，不仅限制其生长，而且还影响其产量及纤维品质，给棉花生产造成极大损失。因此，研究棉花的耐逆分子机制将为利用转基因手段改良棉花抗逆性奠定基础。本论文基于棉花EST公开数据库，鉴定和克隆非生物胁迫应答trihelix-GT基因并解析其生物学功能。2.研究方法与结果通过BLAST分析比对的方法，从棉花EST数据库中获得28个GT基因，命名为GhGT1-26、GhGT29和GhGT30。以陆地棉品种新陆早26号为材料，利用RT-PCR和qPCR分析了28个基因在干旱、盐、冷和ABA处理下的表达谱，确定GhGT7、11、18、23、26、29和30基因为候选基因。通过cDNA末端快速扩增技术（Rapid amplification of cDNA Ends，RACE）克隆这7个基因的全长序列。根据推测的蛋白结构域和保守的氨基酸残基，将这7个基因进行分类：GhGT7和GhGT30蛋白属于GT-2类亚家族，GhGT11和GhGT23属于SIP1，GhGT26属于GT-1，GhGT29属于SH4类，GhGT18推测属于MYB类。qPCR分析表明，7个基因在棉花各组织都有表达，但表达模式不一样。GhGT7基因在叶片中的表达量最高；GhGT11基因在胚珠中的表达量最高，在花中表达量最低；GhGT18基因在胚珠中的表达高于其它组织；GhGT23基因在纤维中的表达量明显高于根、茎、叶、花和胚珠；GhGT26、29、30这3个基因在花和纤维中的表达量最高，在根中的几乎检测不到。通过拟南芥原生质体和洋葱表皮细胞的亚细胞定位分析表明，GhGT7、11、18、23、26、29、30基因编码的蛋白都定位在细胞核内。拟南芥原生质体系统分析GhGT7、18、23、29基因的转录活性表明：GhGT29蛋白具有转录激活活性，GhGT18蛋白具有转录抑制活性，GhGT7和23没有检测到转录激活活性。凝胶阻滞（EMSA）分析发现，GhGT7、11、23和26蛋白既能结合GT元件也能和MYB元件结合，GhGT7蛋白可以结合GT元件GT-1box、GT2-Box、GT-3b和MYB元件MBS1、MRE1、MRE3、MRE4；GhGT11蛋白分别结合Site1、GT-1box、GT2-Box、GT3-Box、GT-3b、MRE3；GhGT23蛋白分别结合Box、Site1、GT-1box、GT2-Box、GT3-Box、GT-3b、MBS1和MRE4；GhGT26蛋白结合Site1、MRE3、MRE4。利用农杆菌介导的转基因技术，将GhGT7、11、18、23、26、29基因分别转入拟南芥中，已分别获得了拟南芥转基因纯系植株，并进行了表达量鉴定，对GhGT11、18、23、26基因的转基因拟南芥植株进行了非生物胁迫下表型分析。转基因植株表型分析表明，在正常生长条件下，过量表达GhGT的转基因拟南芥无论是苗期还是成株期植株，转不同基因株系之间及与野生型之间没有明显差异，在盐和干旱胁迫下，过量表达GhGT23和GhGT26基因的转基因拟南芥植株的耐盐性、耐旱性较野生型Col-0均有明显的提高；而GhGT11和GhGT18转基因植株的耐盐性、耐旱性均降低。对转基因植株根长表型分析发现，无论是在盐和甘露醇胁迫下，转GhGT18和GhGT11基因的植株抑制根的生长，而转GhGT23和GhGT26基因的植株促进根的生长。对ABA处理后的转基因根长分析发现，过表达GhGT18和GhGT26基因植株对ABA没有反应，GhGT23的转基因植株能促进根的生长，而GhGT11基因的转基因植株则抑制根的生长。对正常生长的转基因植株的下游基因分析表明，GhGT23基因通过调控与耐逆性相关的基因DREB1B、COR6.6、COR47、RD22、SAP18、COR15A、DREB2A、DREB2B、STZ、AP2和DREB2C基因的表达，提高转基因植物的耐受性。3.研究结论本论文筛选和克隆了棉花应答非生物胁迫的7个GhGT基因，研究了它们编码蛋白的生化特性；通过转基因植物表明，GhGT11，GhGT18，GhGT23，GhGT26参与了植物的耐逆性调控，并对其调控途径进行了探讨。更多还原

【Abstract】 1. ObjectivePlant growth and productivity are under constant threat from environmental changes in theform of biotic and abiotic stress factors. The most common abiotic stresses are high salinity, lowtemperature and drought, which limit the crop productivity, reducing average yields for mostmajor crop plants by more than50%. In molecular breeding, a key transcription factor cansimultaneously regulate the expression of many functional genes to improve tolerance of crops tovarious stresses. Trihelix transcription factors are a small family only identified in plants. Theycan interact with cis-elements present in the promoter regions of several stress-related genes andregulate their expression to enhance plant stress tolerance. Therefore, trihelix transcription factorsplay a critical role in stress signaling transduction pathways. Cotton is an important crop plant forboth textile and oil, but its growth, yield and fiber quality were affected by various abiotic stresses,such as drought, salinity and low-temperature, doing great harm to cotton production.Understanding the molecular mechanisms of stress tolerance in cotton will lay the foundation forimproving the stress tolerance of cotton by gene manipulation. Based on the EST sequences, wecloned several GT genes responsive to abiotic stresses from cotton and their functions were furtheranalyzed.2. Methods and resultsBy doing BLAST(the Basic Local Alignment Search Tool) search the cotton ESTs database,28GT unigenes, named GhGT1to GhGT26, GhGT29and GhGT30, were obtained. Theexpression profiles of these unigenes under the treatments of drought, salinity, cold and exogenoushormone ABA in Xin-lu-mian26were investigated by both RT-PCR and qPCR method. SevenGhGT unigenes including GhGT7,11,18,23,26,29and30were chosen for further analysis. Thefull-length cDNA of these GhGTs genes were cloned using the method of Rapid amplification ofcDNA Ends (RACE). Based on the deduced protein domains and conserved amino acid residues,these GhGT proteins were classified into different subfamilies: GhGT7and GhGT30belong toGT-2; GhGT11and GhGT23belong to SIP1; GhGT26belongs to GT-1; GhGT29belongs to SH4subfamily and GhGT18belongs to MYB transcription factor family. Although7GhGT genes were ubiquitously expressed in root, stem, leaf, flower, ovule (0DPA) and fiber (12DPA), theirexpression patterns were different. The highest expression level was observed in leaves forGhGT7, in ovules for GhGT18, in fibers for GhGT23and in flowers and fibers for GhGT26,GhGT29and GhGT30. The sub-cellular localization assay in the Arabidopsis protoplasts andonion epidermal cell system revealed that GhGT7,11,18,23,26,29and30proteins werelocalized in the nucleus, indicating that they are nuclear proteins. Using the Arabidopsisprotoplasts assay system, we found that GhGT29protein had trans-activation activity, GhGT18showed trans-repression activity, while no trans-activation activity was detected for GhGT7andGhGT23. Electrophoretic Mobility Shift Assay（EMSA）showed that GhGT7,11,23and26proteins could bind to both GT and MYB elements. Specifically, GhGT7protein could bind toGT-1box, GT2-Box, GT-3b, MBS1, MRE1, MRE3and MRE4; GhGT11protein could bind toSite1, GT-1box, GT2-Box, GT3-Box, GT-3b and MRE3; GhGT23protein could bind to Box,Site1, GT-1box, GT2-Box, GT3-Box, GT-3b, MBS1and MRE4, and GhGT26protein could bindto Site1, MRE3, MRE4, respectively. Employing agrobacterium-mediated transformationtechnique, GhGT7,11,18,23,26and29genes were successfully introduced into Arabidopsisthanliana to generate transgenic plants, respectively. The expression level of transgene wasanalyzed for each homozygous transgenic line. Performance of transgenic plants overexpressingGhGT11,18,23and26genes under abiotic stresses were studied, respectively. Under normalgrowth condition, no visible phenotype difference between wild-type plants and transgenic plantsoverexpressing each GhGT gene was observed. Overexpression of GhGT23and GhGT26significantly enhanced the tolerance of transgenic Arabidopsis plants to high-salinity and droughtstresses compared with wild-type plants, respectively. However transgenic plants overexpressingGhGT11and GhGT18showed sensitivity to high salinity and drought stresses, respectively. Underdrought and high salinity treatments, the performance of root growth of wild-type plants wasbetter than that of transgenic plants overexpressing GhGT18and GhGT11, while the performanceof root growth of transgenic plants overexpressing GhGT23and GhGT26was better than that ofwild-type plants. Compared to wild-type plants, ABA treatment promoted the root growth oftransgenic plants overexpressing GhGT23, but inhibited the root growth of transgenic plantsoverexpressing GhGT11. Transgenic plants overexpressing GhGT18and GhGT26were notsensitive to ABA treatment. Overexpression of GhGT23improved the tolerance of transgenicplants to abiotic stresses probably through up-regulating the expression of downstream genesincluding DREB1B, COR6.6, COR47, RD22, SAP18, COR15A, DREB2A, DREB2B, STZ, AP2andDREB2C.3. ConclusionIn short, seven GhGTs genes responsive to abiotic stresses were identified and cloned, andbiochemical function of their proteins were characterized. Overexpression of each of the four GhGTs genes including GhGT11, GhGT18, GhGT23and GhGT26affected the tolerance oftransgenic plants to abiotic stresses. The possible mechanism of GhGT23-mediated improvementof transgenic plants to abiotic stresses was further discussed.更多还原