【作者】 万丙良；

【导师】 牟同敏； 林拥军；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 植物钙依赖的蛋白激酶（CDPKs）是细胞钙信号的受体，在植物生长发育、病原防御、非生物环境胁迫、离子通道运输等生理反应的信号传递过程中起着重要的调控作用。水稻存在多个CDPKs基因，有少数几个基因的功能比较清楚，它们与水稻的发育或胁迫响应有关，其它大多数基因的功能有待进一步研究。本研究首先分析了水稻CDPKs基因启动子区的胁迫响应元件，然后利用RT-PCR和Northern blot杂交技术并结合水稻表达数据库（RED：http://red.dna.affrc.go.jp/RED/）的数据，对CDPKs基因在不同胁迫环境和不同水稻组织中的转录表达情况进行了分析，根据表达结果鉴定出胁迫响应基因：选取OsCPK8和OsCPK6基因，利用RNAi抑制基因表达技术和超表达技术对它们的功能进行了分析，分别明确了它们在水稻株高的形态建成和非生物胁迫响应中的作用。本研究获得的主要研究结果如下：1水稻CDPKs基因的表达分析1．1通过对CDPKs保守序列的搜索分析，在水稻基因组中鉴定出29个CDPKs基因，它们具有典型的CDPKs结构域，即Ser／The蛋白激酶区、钙调素类似结构域。1．2 29个CDPKs基因启动子区域均含有数量不等的多种调控胁迫响应的顺式元件，暗示这些基因可能与这些胁迫响应有关。这些元件包括ABA响应元件EBOXBNNAPA，光调控元件IBOXCORE、脱水响应元件MYBCORE、MYCATRD22、DRECRTCOREAT，低温响应元件LTRECOREATCOR15，热激响应元件CCAATBOX和防卫反应元件WBOXATNPR1。1．3在水稻表达数据库中检索出11个与水稻CDPKs基因对应的ESTs，它们对低温、干旱、高盐、几丁质激发子处理和稻瘟病病菌侵染等胁迫表现出广泛的转录响应。1．4根据水稻CDPKs基因转录表达的组织特异性，可将水稻CDPKs基因分为7类；新筛选出3个受胁迫诱导发生表达变化的水稻CDPKs基因，OsCPK6、OsCPK17和OsCPK25，其中OsCPK6受干旱和盐胁迫诱导转录表达上升，OsCPK17在低温、干旱和盐胁迫刺激下转录量下降，OsCPK25受热激诱导转录量上升。这些胁迫诱导基因在水稻对逆境胁迫的响应中可能起着重要作用。2 OsCPK8基因功能分析2．1运用RNAi技术构建了OsCPK8基因的抑制表达载体并转化水稻，大部分转基因植株较野生型植株高度降低，植株矮小的原因主要是节间缩短，倒1、2、3节间和穗长的变化对植株高度变化的影响最大。2．2对OsCPK8基因的表达和转基因植株的高度进行共分离检测，结果表明株高与OsCPK8基因的RNA表达量呈现出共分离，矮化植株中OsCPK8基因的表达受到抑制，转录量有不同程度的下降。说明OsCPK8基因表达量的下降是植株变矮的原因。2．3水稻内源赤霉素含量测定表明，OsCPK8 RNAi矮化植株内源GA₃含量显著低于野生型植株的水平；施用外源赤霉素，矮化植株的株高有一定程度的回复，但不能回复到野生型水平；对幼苗进行赤霉素敏感性分析的结果表明，矮化植株对赤霉素不如野生型敏感。这些结果表明OsCPK8 RNAi矮化植株的赤霉素合成和信号传递途径均可能受到阻碍。2．4 OsCPK8 RNAi矮化植株中赤霉素合成相关基因OsGA2ox1和OsGA2ox2，以及赤霉素信号调控基因OsGAI的转录表达发生了改变，在此基础上提出了OsCPK8的信号通路假设。2．5对OsCPK8 RNAi矮化植株进行的细胞学分析表明，矮化植株节间的伸长区细胞纵向长度变短，这可能是植株矮化的细胞学基础。3 OsCPK6基因功能分析3．1利用RT-PCR技术从水稻中分离出OsCPK6基因的全长cDNA片段，根据其核苷酸序列推测的氨基酸肽链包含508个氨基酸，具有典型的钙依赖的蛋白激酶结构。OsCPK6定位于细胞膜和细胞核。3．2将OsCPK6全长cDNA片段置于CaMV35S启动子的调控下导入水稻，获得16个单拷贝转基因植株，RNA水平的表达分析表明，转基因植株中OsCPK6基因转录量比非转化对照高。3．3干旱和盐胁迫试验表明，3个转OsCPK6基因株系S1、S3和S5在胁迫下的萎蔫苗率明显低于非转化对照，说明OsCPK6超表达转基因水稻对干旱和盐胁迫的耐性增强。3．4对转OsCPK6基因水稻进行生理分析表明，在胁迫环境下转基因水稻叶片中脯氨酸含量较非转化对照植株高，这可能是转OsCPK6基因水稻抗逆性增强的生理原因。3．5转基因水稻中OsCPK6的超表达使得胁迫诱导转录因子基因SNAC1的转录表达增强，OsCPK6可能通过SNAC1来调控其它胁迫响应基因参与水稻对干旱等逆境胁迫的响应。更多还原

【Abstract】 CDPKs take important roles in calcium-mediated signal transduction involved in plant growth and development, defenses to pathogen, responses to abiotic stresses, ionic transportation in channels. In rice, functions of most CDPKs genes are elusive with the exception of several CDPKs genes that have been known to take roles in development and responses to stresses. In the research, we identified firstly the cis-elements in responsive to stresses in promoter regions of rice CDPKs genes, then analyzed the transcription expression of CDPKs genes under different stress conditions and in different rice tissues according to the data from rice expression database （RED: http://red.dna.affrc.go.jp/RED/） and the results of RT-PCR and Northern blot. Based on expression analysis, CDPKs genes were classified and stress-responsive CDPKs genes were identified. Lastly, the functions of 0sCPK6 and 0sCPK8 were analyzed with RNAi technology and gene overexpression technology.The main results of this research are as follows:1 Transcription expression analysis of rice CDPKs genes1.1 29 rice CDPKs genes were identified in rice genome through sequence comparing with conserved domain of plant CDPKs genes; the putative amino sequences have typical CDPKs domains of Ser/Thr kinase domain and CaM-like domain.1.2 The promoter regions of CDPKs genes contain stress-responsive cis-elements of EBOXBNNAPA, IBOXCORE, MYBCORE, MYCATRD22, DRECRTCOREAT, LTREC0REATC0R15, CCAATBOX and WB0XATNPR1, which suggested that rice CDPKs genes might be in responsive to stress stimuli.1.3 11 ESTs corresponding to 11 rice CDPKs genes in rice expression database were widely in responsive to the stresses of cold, drought, salt, chitin and infection of rice blast pathogen.1.4 CDPKs genes were classified as 7 groups on the base of tissue-specific expression of CDPKs genes, and 3 CDPKs genes of 0sCPK6, OsCPK17 and OsCPK25 were firstly identified to respond to cold, drought, salt stresses and heat shock.2 The function analysis of 0sCPK82.1 RNAi technology was performed to inhibit the expression of 0sCPK8, transgenic rice plants with down-regulated expression of 0sCPK8 were obtained, the plant height of transgenic plants were shorter than that of the wild type.2.2 The plant height of transgenic plants co-segregated with transcription levels of OsCPK8 in transgenic plants, dwarf plants had low transcriptions of 0sCPK8. This result indicated that the down-regulated expression of 0sCPK8 resulted in the shortened culms of transgenic plants.2.3 The GA₃ contents in dwarf transgenic plants were lower that in WT; spraying GA₃ on dwarf plants could partially recover plant height of dwarf plants but not to the height of WT; the seedlings of dwarf plants were less sensitive to exogenous GA₃ than WT. These results indicated that the synthesis of GA and GA signal pathway were inhibited to some extent.2.4 Based on the changes of transcription of OsGA2ox1, OsGA2ox2 and OsGAI resulted from the down-regulated transcription of 0sCPK8 in dwarf plants, a putative signal pathway of OsCPK8 was presented in this paper.3 The function analysis of 0sCPK63.1 The full-length cDNA of OsCPK6 was isolated and sequenced, and the putative amino sequence had a Ser/Thr kinase domain and an EF-hand domain. OsCPK6 was located in cell membrane and nucleus.3.2 OsCPK6 was transformed into rice variety, Wuyujing 3, and 16 transgenic rice plants with single copy of exogenous gene were obtained, OsCPK6 had increased transcription in transgenic rice plants.3.3 Three transgenic rice lines of S1, S3 and S5 showed that the tolerance to drought and salt stresses increased and the rates of wilted seedling under stresses of drought and salt were lower than non-transgenic rice.3.4 Transgenic rice had higher content of proline in leaves under drought and salt stresses than negative transgenic controls. This may be physiological mechanism of increased tolerance to environmental stresses of OsCPK6 transgenic rice.3.5 The transcription of the stress-inducible transcription element gene SNAC1 was up-regulated by OsCPK6 overexpression in transgenic rice. OsCPK6 may participate in responses of rice to environmental stresses through regulating SNAC1.更多还原