【作者】 李新征；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 棉花是较耐盐碱的作物之一,对改良和有效利用盐渍地具有重要意义。但棉花的耐盐能力有限,要在盐碱地广泛种植,必须进一步提高棉花的耐盐能力。传统的常规育种方法无法大幅度提高棉花的耐盐性,分子生物学的发展,为大幅度提高棉花的耐盐性提供了新的方法。但要通过转基因技术提高棉花的耐盐性,首先要了解棉花耐盐的分子机制,并解决基因来源问题。而这两个问题解决的基础首先是要克隆到足够多的与棉花耐盐相关的基因。而目前大规模克隆与耐盐相关基因的最有效方法之一就是cDNA文库筛选法。本研究以耐盐棉花品种中棉19号为材料,构建了棉花盐胁迫诱导cDNA文库,对文库的筛选鉴定表明,构建的棉花子叶cDNA文库质量较好。通过反相Northern差异筛选法获得17个与盐胁迫相关的新基因。并选择5号、17号、21号和30号克隆进行功能研究分析。研究发现5号和17号克隆属于棉花金属硫蛋白基因家族成员,30号克隆是PPIC型(PPIC- PPIASE-2)-肽酰脯氨酰顺反异构酶(parvulin-type peptidyl-prolyl cis/trans isomerase),而21号克隆可能是一种紫外线B抑制蛋白。金属硫蛋白(Metallothionein,MT)是一类低分子量(6000～9000Da)、高Cys含量、具有金属结合能力的多肽,它具有独特的氨基酸排列顺序,即多肽的N端和C端具有两个富含Cys的金属结合结构域,其中的Cys按CXnC的方式排列。MT基因和蛋白广泛存在于动物、植物、真菌、绿藻中。植物金属硫蛋白按结构特点分为三类。5号克隆编码的GhMT1与17号克隆编码的GhMT2为棉花I类金属硫蛋白。对这两个基因进行序列比较、表达分析和功能鉴定。主要结果如下:1. 5号克隆和17号克隆的序列同源性分析表明,二者的核苷酸序列的同源性为91.15%,氨基酸同源性为85.94%,可以断定二者为同一棉花基因家族成员。该基因家族编码蛋白与覆盆子、葡萄、中华猕猴桃、番木瓜等许多植物中的I类金属硫蛋白的同源性为70.1%,具有典型的植物I类金属硫蛋白序列特点,聚类分析表明该棉花基因家族与覆盆子、葡萄、中华猕猴桃、番木瓜等植物的I类金属硫蛋白具有较近的亲缘关系。因此,可以断定二者属于植物I类金属硫蛋白,命名为GhMT1、GhMT2。2. Northern杂交分析表明GhMT1受NaCl胁迫诱导表达,在一定的时间范围内其表达水平随NaCl诱导时间的延长而增加,但是在盐胁迫条件下不能被快速诱导。GhMT1也能被CuSO4、ZnSO4、低温、干旱、ABA和乙烯诱导,说明GhMT1具有金属硫蛋白受重金属诱导的功能,也说明对盐胁迫的非专一性,而是受各种非生物胁迫的诱导。同时暗示着GhMT1受ABA和乙烯信号途径的调控,可能是ABA、乙烯信号途径的靶基因。3. Northern表达分析还表明在抗氧化剂N-乙酰基-L-半胱氨酸(N-Acetyl-L-cysteine,NAC)存在的条件下,GhMT1不受NaCl、低温、干旱、百草枯(PQ,paraquat)的诱导。对过氧化氢相对水平的测定结果表明,NAC有效的抑制了NaCl、低温、干旱胁迫条件下过氧化氢水平。说明活性氧自由基是非生物胁迫诱导GhMT1及其同源家族表达的重要信号物质。4.GhMT1和GhMT2在转基因烟草中过量表达,用GhMT1和GhMT2特异探针进行检测,Northern杂交结果说明GhMT1和GhMT2只在转基因植株中表达。同野生型相比,超表达GhMT1和GhMT2的转基因烟草在200mM NaCl、300mM NaCl条件下以及4℃条件下与25%PEG条件下较好的生长。说明GhMT1及其同源家族能提高植物的抗盐能力,也能提高植物的抗其它主要非生物胁迫的能力。5.对转基因烟草生理指标测定结果表明,在盐、4℃、25%PEG胁迫条件下转基因烟草SOD酶活性明显高于野生型烟草。说明GhMT1能够提高植物清除活性氧自由基能力,从而提高植物抗盐、抗低温及抗干旱等非生物胁迫的能力。6.对GhMT1体外结合Zn2+能力的测定表明,GhMT1具有金属硫蛋白家族能够结合金属离子的特性,每一分子GhMT1最多能结合5个锌离子。肽酰脯氨酰顺反异构酶(peptidyl proline cis/trans isomerase,PPIase)是一种催化肽酰-脯氨酰的肽键顺反异构的一类酶,在进化上高度保守,成员多样,并且分布广泛,在生物体的生理过程中发挥重要的作用。脯氨酰异构酶超家族可以分为FK506结合蛋白(FK506-binding proteins,FKBPs)、亲环素(cyclophilins,Cyp)和Parvulins三个独立的蛋白家族,每个家族又有众多成员。30号克隆编码的GhPPI是Parvulins家族中的肽酰脯氨酰顺反异构酶,对这个基因进行序列比较、表达分析和功能鉴定。主要结果如下:1.序列同源比较分析表明,棉花GhPPI具有PPIC- PPIASE-2(PPIC型-肽酰脯氨酰顺反异构酶)保守区,是parvulin家族顺反异构酶中的成员,酶学分类为(EC 5.2.1.8)。同源蛋白聚类分析表明GhPPI和拟南芥AtPIN2以及水稻中的肽酰脯氨酰顺反异构酶具有高达92%以上的同源性,与人的Par14和Par17也有高达55%的同源性。棉花GhPPI除具有顺反异构酶保守区外,N端还具有富含Lys的碱性氨基酸区。通过综合比较分析表明,棉花GhPPI可能是parvulin家族人Par17类型成员。2.跨膜区分析表明,棉花GhPPI没有跨膜区。GhPPI-GFP融合蛋白洋葱表皮亚细胞定位表明,GhPPI主要定位于细胞核中,并与染色体结合。核定位信号分析表明,没有明显的核定位信号。前人研究表明Par14和Par17的核定位信号位于N端富含Lys的碱性氨基酸区,并不剪切去,并且N端的碱性氨基酸区是结合DNA所必需的。推测GhPPI与Par14和Par17具有高度同源的N端富含碱性氨基酸区也具有相似功能。3.通过原核表达纯化到了棉花GhPPI蛋白,选用N-Succingl-Ala-Ala-Pro-Phe-4- nitroanilides底物,对棉花GhPPI蛋白顺反异构酶活性进行分析测定。结果表明棉花GhPPI蛋白具有较低的顺反异构酶活性。棉花GhPPI蛋白是植物中第一个鉴定的具有顺反异构酶活性的parvulin家族中的成员。4.用Northern杂交技术进一步分析棉花GhPPI的内源表达情况。结果显示,GhPPI有一定的本底表达水平,受300mM NaCl诱导后,其表达水平升高,并在一定的时间范围内随NaCl诱导时间的延长而增加。说明GhPPI顺反异构酶活性与植物抗胁迫有一定的关系。5.前人将AtPIN2分类到parvulin家族人hPar14类型中,通过对我们实验结果分析讨论发现,AtPIN2与GhPPI同源性高达93%,将两者归类到的parvulin家族人hPar17类型更科学。GhPPI的许多研究结果都暗示GhPPI与hPar17功能类似,推测GhPPI可能也具有hPar17的相似功能。6.从拟南芥中克隆到棉花GhPPI在拟南芥中的同源基因AtPIN2基因,构建了AtPIN2基因的超表达、反义表达载体和RNAi干涉载体,并获得相应的转基因植株。发现AtPIN2基因RNAi干涉的T0代拟南芥幼苗生长迅速。转基因拟南芥植株的获得,为通过采用反向遗传学方法研究AtPIN2和GhPPI等parvulin家族基因功能创造了条件。高等植物的光受体可分为4种类型:光敏色素(phytochrome)、隐花色素(cryptochrome)、趋光素(phototropin)和超级色素(superchrome)。其中,隐花色素和趋光素都能吸收蓝光,属于蓝光受体。21号克隆GhUVB1可能编码一种紫外线B抑制蛋白。对GhUVB1进行初步的结构与功能鉴定分析,结果如下:1.氨基酸序列多重对比分析表明棉花GhUVB1编码蛋白在第60个氨基酸到第114个氨基酸的区域内具有“aaaxxxxmxxpxxaxaaxxxxxpslknfllsixxggxvxxxixgxvxxvsnfdpvk”高度保守区域,与之同源性较高的蛋白多数属于紫外线B抑制蛋白。进一步同源分析比较表明,棉花GhUVB1与光系统II的X亚基(PsbX类似蛋白)也有一定的同源性,也可能是PsbX类似蛋白。2.跨膜区分析表明,棉花GhUVB1没有跨膜区。GhUVB1-GFP绿色荧光融合蛋白洋葱内表皮亚细胞定位研究发现,GhUVB1主要定位于细胞膜上,而细胞核等部位也发现有绿色荧光的存在,但分布不均匀,推测GhUVB1也定位于细胞核膜上。这与GhUVB1蛋白的跨膜区理论分析相吻合。3.构建了棉花GhUVB1蛋白的超表达载体,并转化到拟南芥中超表达,结果发现超表达棉花GhUVB1蛋白的拟南芥T0代植株,与野生型相比,植株明显矮小,且开花期平均延迟7天左右。这一重要异常现象为下一步揭示棉花GhUVB1蛋白功能提供了重要线索。4.棉花GhUVB1蛋白定位于质膜与核膜或核内的,理论上是与紫外光信号接受与传递密切相关的蛋白,超表达该蛋白的转基因拟南芥植株出现生长延迟现象,这一现象与隐花色素CRY1的表现相似。推测棉花GhUVB1可能是一种隐花色素或与隐花色素密切相关的蛋白。更多还原

【Abstract】 Cotton is one of the most important economic crops in our country. People are always interested in identifying and developing new cotton breed to make it can be widely cultivated in saline-alkali soil. Recently, a great achievement has been obtained. However, the salt tolerance ability of cotton is still very limited. So identification of the corresponding salt-tolerant cotton will have important implications for agriculture. The transgenic technology provides us with new ways to improve the plants tolerance by transferring useful genes to different plants. In this experiment, a NaCl-induced cDNA library of cotton seedlings was constructed in order to isolate more salt-tolerance genes from cotton. The library was validated to be successful by titering. 17 salt-induced clones were identified after screening the cDNA Library. Clone 5, 17, 30, and 21 were selected to studied respectively.Metallothioneins (MTs) are defined as a class of proteins with the characteristics of low molecular weight, high cysteine (Cys) residue content and metal-binding ability. They have been widely found in animals, plants, fungi and cyanobacteria. Plant MT-like proteins are divided into three major classes (MT-I, MT-II, MT-III ), which are distinguishable on the basis of the distribution of cysteine residues in their amino acid sequences. Clone 5 and 17 belong to class MT-I. The stucture and function of Clone 5 and 17 were studied. The main results are as follows:1. Clone 5 and 17 have high sequence homology with each other, the identity of Nucleotide sequences is 91.15%, and identity of the amino acid residues sequences can reach 85.94%. These two clones showed high homology with metallothein-like genes from various plants species, such as Rubus idaeus, Carica papaya, Vitis vinifera, Actinidia chinensis etc. The phylogenetic analysis of these proteins suggested that those proteins all belong to the same protein family. Take together, our results indicated that clone 5 and 17 encode two MT-I proteins, which were designated as GhMT1 and GhMT2.2. Northern blot analysis indicated that the mRNA accumulation of GhMT1 was induced by salt stress (300mM NaCl), and the mRNA level kept increasing in the following 12 hours. The expression of GhMT1 was also up-regulated by 300μM CuSO4, 300μM ZnSO4, low temperature (4℃), drought (25% PEG), ABA (abscisic acid) and ethylene, suggesting that MT-I in cotton was not a specific salt stress response gene family but an abiotic stress response gene family. This result also suggested that GhMT1 might play a role in signal transduction of ABA and ethylene.3. Northern blot analysis also showed that the expression was repressed by N-Acetyl-L-cysteine (NAC) when treated by NaCl, low temperature (4℃) or drought (25%PEG). Analysis of H2O2 level showed that NAC repress the H2O2 level increase in above stress. .Therefore, reactive oxygen species (ROS) is essential for activating the GhMT1 and other member expression of GhMT I family under abiotic stress.4. The transgenic tobacco plants overexpressing GhMT1 and GhMT2 grew better than wild type tobacco in 200 and 300 mM NaCl solution. The transgenic tobacoo plants also grew better under low temperature (4℃), drought (25%PEG) condition. Northern blot analysis showed that the GhMT1 and GhMT2 genes had higher transcription levels in transgenic tobacco plants, while there was no GhMT1 and GhMT2 expression in wild-type tobacco plants.5. The increased SOD activities were observed in transgenic tobacco plants and wild type tobacco plants after various abiotic treatments (NaCl, 4℃, 25% PEG). However, the SOD activities in transgenic plants was higher than in wild type plants, indicating that over-expression of GhMT1 could increase the activity of SOD, which maybe the reason that GhMT1 and its members could improve abiotic stress tolerance of cotton.6. To test the binding of GhMT1 to metal Zn2+, we expressed GhMT1 in E. coli. The results showed that the purified GhMT1 could bind Zn2+ions in vitro ,and the binding ability is no more than five Zn2+ions per MT molecule.Peptidyl prolyl cis/trans isomerases (PPIases, EC 5.2.1.8) play a role in protein folding and function by twisting the backbone of target proteins. PPIases are abundantly expressed in virtually all organisms and all cellular compartments .These ubiquitous proteins can be divided into three families, cyclophilins, FK506-binding proteins (FKBP), and parvulins. PPIases may also act as chaperones either in a PPIase domain-dependent or-independent manner, and they may have essential overlapping functions with other PPIases and chaperones. GhPPI of clone 30 belong to parvulins. The stucture and function of GhPPI were studied. The main results are as follows:1. Clone 30 has high sequence homology with peptidyl-prolyl cis-trans isomerase PPIC-type family (Pin4) from Arabidopsis Thaliana , Oryza sativa and Mus musculus etc. The phylogenetic analysis of these proteins suggested those proteins belong to parvulins family. Sequence alignment showed that GhPPI is more similar to hPar14 or hPar17 type parvulin with 55% identity to hPar14(also known as PIN4/EPVH/hPar14) and hPar17. Taked together, our results indicated that clone 30 encodes peptidyl-prolyl cis-trans isomerase, which is more similar to hPar17, and were designated as GhPPI.2. Subcellular localization of the transiently expressed pBI121-GhPPI-GFP fusion protein in onion epidermal cells was done, the results show GhPPI-GFP fluorescence is concentrated to the nucleus or to the chromosome.3. To assay activity of GhPPI PPIase, expression vector of pET30 a (+)-GhPPI was Constructed and expression of pET30 a (+)-GhPPI was induced with IPTG. The results showed that the purified GhPPI has low activity, these indiced the GhPPI is a new peptidyl-prolyl cis-trans isomerase.4. Northern blot analysis indicated that the mRNA accumulation of GhPPI was induced by salt stress (300mM NaCl), and the mRNA level kept increasing in the following 12 hours. The result suggested that GhPPI might play a role to improved abiotic stress tolerance of cotton.5. GhPPI share a similar C-terminal PPIase domain and an similar N-terminal Lys-rich domain with hPar14 and hPar17. The basic N-terminal domain of hPar14 or hPar17 is responsible for the phosphorylation regulated partition between cytosol and nucleus and its affinity for DNA binding. So GhPPI may have regulatory functions as that in hPar14 and hPar17. It may be better that AtPIN, as GhPPI, also be classified into hPar17 type than into hPar14 before.6. AtPPI was cloned and over expression vector, antisense expression vector and RNAi pFGC5941 vector were constructed. The respective transgenic Arabidopsis plants were identified. 14 days old of Seedlings of transgenic RNAi Arabidopsis were grown stronger and faster than wild type, this is an interesting phenomena.The sequence and expression analysis, function identification of Clone 21 were also studied. The main results are as follows:1. Clone 21 has high sequence homology with ultraviolet-B-repressible protein. The phylogenetic analysis of these proteins suggested those proteins belong to ultraviolet- B- repressible protein family. Clone 21 has also high sequence homology with photosystem II subunit X(Psb X). Take together, our results indicated that clone 21 encodes putative utative ultraviolet-B-repressible protein, which were designated as GhUVB1.2. Subcellular localization of the transiently expressed pBI121-GhUVB1-GFP fusion protein in onion epidermal cells shows GhPPI-GFP fluorescence is concentrated to the membrane of cell and karyotheca repectively.3. Over expression of GhUVB1 in the transgenic Arabidopsis plants show that the T0 transgenic Arabidopsis plants were grown slowly and flower lag of 7days. The phenomic lag of transgenic plant by over expression GhUVB1 in Arabidopsis plants show was smilar to cryptochrome CRY1. Take together, these results indicated that GhUVB1 maybe is closed relative with cryptochrome.更多还原