【作者】 胡红红；

【导师】 熊立仲； 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 干旱、冷害和盐碱等非生物逆境对于作物的产量和品质有着非常重要的影响。干旱是植物逆境最普遍的形式，在许多地区是农业发展的瓶颈。因而抗逆育种特别是抗旱育种一直受到重视。虽然有很多报道认为超量表达某些基因可以提高转基因植株的抗逆性，但真正用于大田实验或在作物中尝试的少之又少。本研究以最终获得用于水稻抗逆遗传改良的基因为目的，通过超量表达一些对逆境起核心调控作用的转录因子，系统地对这些转基因水稻在大田和温室环境中进行抗逆性分析和鉴定，初步鉴定出SNAC1和SNAC2等基因在抗逆遗传改良中的有效性，并对这两个基因进行了深入的功能研究和抗逆性机制探讨。主要结果如下：1．在载体pCAMBIA1301S(含double CaMV 35S启动子)，pCAMBIA1301A(含Actin1启动子)，pCAMBIA1301U(含Ubiquitinl启动子)和pCAMBIA1301H(含HVA1-like干旱诱导启动子)的基础上，构建了43个目标基因超量表达载体和1个诱导表达载体；在载体pHELLSGATE2的基础上，构建了32个目标基因的RNA干扰(RNAi)抑制表达载体。2．通过northern和Southern杂交对转基因植株进行了表达量的检测和转基因拷贝数的检测，结果表明大概有50％左右的植株为超量表达，30％左右的植株为单拷贝转基因插入。3．通过田间对苗期转基因水稻的干旱筛选，筛选出4个目标基因的转基因家系干旱敏感和5个目标基因的转基因家系表现为抗旱增强。4．通过田间对成株期转基因水稻的干旱筛选，筛选出4个目标基因的转基因部分家系抗旱性明显增强。综合抗旱筛选结果，详细鉴定了SNAC1的功能；同时对另外两个基因(SNAC2和T052)的功能进行初步分析。5．对SNAC1基因的表达谱分析，表明SNAC1受干旱、高盐、低温和ABA的诱导表达，并且在干旱条件下，SNAC1在保卫细胞特异诱导表达。6．酵母细胞中的反式激活和单杂交实验以及亚细胞定位结果表明SNAC1具有NAC(NAM，ATAF and CUC2)转录因子的特性，其中241-271位的氨基酸序列对于SNAC1具有转录激活功能是必不可少的，同时SNAC1能结合具有CACG和CATGTG核心序列(类似于拟南芥中鉴定的NACRS序列)的顺式作用元件，并促进报告基因的表达。7．SNAC1超量表达转基因植株在成株期和苗期的抗旱性明显增强。特别是在重度干旱条件下，野生型植株的结实率低于5％，而转基因水稻的结实率在20％以上。但是转基因与野生型植株的表型没有任何差异。8．扫描电镜观察结果表明转基因植株的气孔关闭数要显著高于野生型植株；在干旱过程中，转基因植株丧失水分的速率要慢于野生型，具有更低的临界相对含水量。9．无论是个体水平还是细胞水平，SNAC1的超量表达能提高转基因水稻的耐盐性。10．SNAC1的超量表达引起转基因水稻植株的ABA敏感性。11．SNAC1超量表达的转基因水稻植株进行基因表达谱分析，发现有很多与气孔运动、渗透调节、细胞膜稳定性、脱毒作用等相关的基因都上升表达。12．T052基因(编码MYB类转录因子)也受非生物逆境的诱导表达，但在水稻和早稻之间存在表达量的差异；与GFP的融合基因的表达谱分析表明T052也在保卫细胞中表达。13．SNAC1蛋白与T052启动子的互作，它们在保卫细胞的表达模式以及SNAC1转基因植株的基因表达谱分析等结果表明T052可能是SNAC1的下游调控基因，SNAC1和T052在调控气孔关闭中可能起着重要作用。14．SNAC2也具有转录激活、DNA结合和定位在细胞核等转录因子所通常具有的特性。它不仅受非生物逆境(干旱、高盐、低温和ABA)的诱导表达，同时还受JA和伤害的诱导表达。15．SNAC2在水稻中超量表达或诱导表达后能提高转基因植株的耐盐性和耐冷性，同时也大大提高了对ABA的敏感性。16．考虑到SNAC1的潜在应用价值，本研究进一步构建了两个应用型的遗传转化载体pSMDP01和pSMDP01。载体中将SNAC1作为筛选标记基因，利用具有双向诱导活性的启动子OCPI1P来驱动筛选标记基因和抗逆基因(或同时控制多个抗逆基因)的同时表达。更多还原

【Abstract】 Drought and salinity are major abiotic stresses reducing crop production and qualityand are critical bottlenecks to the development of agriculture in many countries.Therefore, stress resistance has long been targeted in designing better crops. Althoughmany reports have shown that over-expression of some stress-inducible genes intransgenic plants can improve drought and salt resistance, very few are conducted ineconomically improtant crops or tested under the field conditions.The objective of this research is to isolate and characterize some transcriptionfactors conferring drought resistance and to utilize them for genetic improvement ofstress resistance in rice. In this study, large number of stress-inducible transcription factorgenes were over-expressed in rice and transgenic rice were tested for drought resistanceat both seedling and anthesis stages in the field. Based on the evaluation of droughtresistance and functional characterization of a few genes, we found that the SNAC1 andSNAC2 genes showed very significant effect in improving drought resistance and salttolerance in rice. The main results are as follows:1. A total of 33 genes were constructed for over-expression, and 31 genes wereconstructed for RNAi suppression.2. Expression level and copy number of T-DNA were analyzed by northem andSouthern blotting respectively. Results showed that 50% of transgenic plants hadover-expression of transgenes and 30% of transgenic plants had one copy of transgene.3. Compared to the wild type (WT), transgenic seedlings of four target genesshowed drought sensitivity and of five genes showed drought resistance when tested inthe nursery field of rice.4. Transgenic rice of four genes showed increased drought toletance when droughttesting was conducted in the field and PVC pipes at the anthesis stage. According to, theresult of drought testing, we chose SNAC1 and SNAC2 for detail analysis of theirfunctions in drought resistance and/or salt tolerance.5. Northern blot analysis revealed that SNAC1 could be induced in leaves by drought,salt, low temperature, ABA and other stresses. Under drought stress, SNAC1 was predominantly induced in guard cells.6. Transactivation assay, yeast one-hybrid assay, and subcellular localizationanalysis showed that SNAC1 functions as a transcription factor belong to NAC (NAM,ATAF and CUC2) family. The region of amino acid 241-271 is essential to transcriptionalactivation activity. Meanwhile, SNAC1 can bind to the NACRS-like sequence containingCACG and CATGTG core sequences identified in Arabidopsis.7. SNAC1-overexpressing transgenic rice showed increased drought resistance atboth seedling and anthesis stages. Under severe drought stress, seed-setting rate oftransgenic lines could remain more than 20% while that of WT was less than 5%. Nosignificant difference of morphological and agronomic traits was observed betweentransgenic and WT plants.8. SEM analysis indicated that SNAC1-overexpressing transgenic plants have morestomatal closed than WT under normal and drought conditions. Under drought stress,transgenic plants showed delayed leaf-rolling, lost water more slowly, and had lowerminimum relative water content (mRWC) for re-establishing turgor.9. Over-expression of SNAC1 significantly enhanced salt tolerance of transgenic riceat both individual and cellular levels.10. Over-expression of SNAC1 enhanced ABA sensitivity of transgenic plants.11. Many genes related to stomatal movement, osmotic adjustment, cell membranestability and detoxification were up-regulated in the SNAC1-overexpressing transgenicplants.12. T052, encoding a MYB protein, was up-regualted in the SNAC1-overexpressingtransgenic plants. This gene was also induced by abiotic stresses, but the induction levelshowed difference between irrigated rice and upland rice cultivars. Moreover, this genewas expressed in guard cells under drought stress conditions.13. Results of the interaction between SNAC1 and T052 promoter, inducedexpression of both SNAC1 and T052 in guard cells, and increased expression of T052 inSNAC1-overexpressors, all together indicated that T052 was a target gene of SNAC1 mayinvolve in regulation of stomatal closure.14. SNAC2 was induced not only by abiotic stresses (such as drought, high salinity, low temperature and ABA), but also by biotic stress (such as wounding and jasmonicacid). It also functions as a transcription factor belonging to NAC family.15. Over-expression of SNAC2 enhanced salt and cold tolerance and ABA sensitivityof transgenic plants.16. Considering the potential usefulness of SNAC1 in gene engineering for stressresistance improvement, two vectors (pSMDP01 and pSMDP02) for genetictransformation were constructed. SNAC1 was used as a marker gene and OCPI1 promoterwith a bidirectional stress-inducible activity was used to control the expression of boththe marker gene and target gene simultaneously.更多还原