【作者】 刘少伟；

【导师】 徐朗莱；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 类枯草杆菌蛋白酶(Subtilases)在植物体内广泛分布,且数目众多,广泛参与植物的生长发育,以及响应外界环境变化参与代谢调控等,但目前该类蛋白酶在植物体内的具体功能和作用机制等方面还不完全清楚。本课题组在前期研究中,从小麦暗诱导衰老的叶片中分离纯化了一种与叶片衰老相关的类枯草杆菌丝氨酸蛋白酶,命名为TaSSP1(Triticum aestivum Subtilisin-like Serine Protease1),并通过质谱鉴定所得数据在NCBI数据库中比对,获得了该蛋白酶氨基酸序列的部分信息,但该蛋白酶在小麦生长发育中到底具有哪些功能不完全清楚。本研究根据已知的该蛋白酶基酸部分序列,采用RACE的方法得到了小麦的该蛋白酶基因TaSSP1全长序列。之后,通过将TaSSP1基酸序列在国际蛋白酶数据库(MEROPS)中进行比对,发现了在拟南芥中与该蛋白酶相似度达77.8%的同源蛋白AT3G14067,而AT3G14067蛋白在拟南芥中的具体功能也尚不清楚。因此,本文在上述研究结果的基础上,进一步通过RT-PCR、荧光定量PCR、Western blot、SDS-PAGE、天然梯度凝胶活性电泳和转基因技术等方法手段,主要从基因的转录和蛋白表达及酶活性表现的水平上研究分析了TaSSP1和AT3G14067基因在小麦和拟南芥生长发育及在响应环境变化中的具体功能,得到的主要研究结果如下：1)根据已知TaSSP1的氨基酸部分序列,采用RACE的方法获得了小麦蛋白酶基因TaSSP1全长序列,并在GenBank进行了登录,登记号为：JX962746。TaSSP1基因CDS区全长2361bp,编码786个氨基酸,在丝氨酸蛋白酶家族中属于S8A家族。对TaSSP1蛋白结构进行生物信息学分析,该蛋白酶含有五个结构域,分别为N端信号肽、19Inhibitor区域、PA (Protease-associated)结构域、催化结构域和C端延长区域。通过荧光定量PCR对jTaSSP1基因的小麦组织特异性表达情况进行分析,发现TaSSP1基因在不同衰老程度的叶片中,衰老程度更严重的叶片中TaSSP1的mRNA表达量更高；在叶片的不同部位中,叶尖部位的TaSSP1表达量较高；暗处理叶片衰老中,发现随着暗处理时间延长,叶片衰老程度加深,叶片TaSSP1的mRNA表达水平和蛋白酶活力均逐渐升高。叶片TaSSPl基因的表达亦受植物激素ABA、6-BA和乙烯的影响,其1nRNA表达和酶活力水平随着ABA处理时间(0-48h,或0-72h)的延长逐渐不断升高；而在6-BA处理下,其mRNA表达和酶活力水平都是呈现先升高后降低并降低到对照(0h)时的以下水平。结果还发现,TnSSP1基因的表达还能明显地被外界渗透胁迫所诱导,如小麦在200mMNaCl、蔗糖和甘露醇以及16%PEG6000的胁迫处理下,叶片中TnSSP1基因的mRNA表达量均明显上升。Western blot与SDS-PAGE实验结果显示,在ABA和16%PEG6000处理的72h过程中,随着TaSSP1蛋白表达水平的不断提高,叶片Rubisco大亚基的含量逐渐减低,表明随着TaSSP1蛋白酶含量的不断升高,Rubisco大亚基的降解程度逐渐加剧。而在6-BA处理下,其蛋白表达水平也是呈现先升后降并降低到低于对照(0h)时以下水平,Rubisco大亚基的含量几乎没有发生明显的变化。另外,实验结果还发现：Cu2+和Ca2+能显著促进TaSSP1酶活力的提高,Zn2+和Mn2+对该酶也有一定的促进作用,但Mg2+和Co2+则对酶活力没有促进作用。以上结果表明：TaSSP1可能是小麦生长发育中的一种重要的Ca2+依赖性的类枯草杆菌丝氨酸蛋白酶,该蛋白酶不仅密切与小麦的衰老进程相关,而且能通过上调基因表达及加快蛋白质周转去积极应对如渗透胁迫等的环境胁迫。2)对AT3G14067蛋白酶进行生物信息学的结构分析,发现AT3G14067蛋白酶与TaSSP1蛋白酶的结构非常相似,同样属于S8A蛋白家族,亦含有N端信号肽、19Inhibitor区域、PA结构域、催化结构域和C端延长区域等五个结构域。通过半定量RT-PCR对AT3G14067基因在拟南芥的组织特异性表达进行分析,结果显示：AT3G14067基因在茎、莲座叶和茎生叶中的mRNA表达量最高,这与在植物膜蛋白数据库中比对分析的结果相吻合。研究结果还显示,AT3G14067基因能明显受植物激素ABA、6-BA、乙烯和水杨酸的诱导而上调表达,生长素类似物NAA和赤霉素可轻微提高AT3G14067基因的表达水平,但在茉莉酸甲酯处理下,AT3G14067的转录表达水平没变化。此外,AT3G14067同样能明显受NaCl、甘露醇、PEG6000和蔗糖等渗透胁迫的诱导而上调表达。暗诱导处理拟南芥莲座叶叶片后,发现处理12h后AT3G14067基因的转录表达水平开始上调,48h时的表达量达到最高；在不同衰老程度的莲座叶叶片中,发现衰老程度严重的叶片中AT3G14067基因的转录表达水平更高,表明AT3G14067基因也与叶片的衰老相关。在野生型拟南芥种子的萌发实验中,发现在黑暗、蓝光、远红光的培养条件下,AT3G14067基因的mRNA表达量会显著上升,其中以黑暗和远红光处理的最为明显。这表明AT3G14067基因对不同光质也有响应。上述结果初步表明,AT3G14067蛋白酶也不仅与拟南芥的衰老有关,而且可能与拟南芥的生长发育密切相关。3)通过转基因获得了AT3G14067基因过表达拟南芥株系OX-3、OX-4和OX-6,经荧光定量PCR鉴定突变体ssp-1为纯合体。对拟南芥的野生型、突变体和过表达株系的表型进行比较发现,在黑暗萌发条件下,AT3G14067基因过表达株系下胚轴显著伸长,突变体株系轻微伸长,突变体与野生型的差异不明显；过表达株系抽苔时间与野生型的相比要提前5天左右,但过表达植株和突变体植株的种子均会提前成熟；在自然生长条件下,过表达植株还表现出主茎顶端生长点丢失,株高变矮,而突变体则株高变高,表型相反；过表达株系植株的分枝数、果荚数显著高于突变体和野生型植株,但过表达株系果荚长度变短、单果荚种子数变少、单果荚重量减轻、单株种子总数提高,而突变体则表现为果荚变长、单果荚种子数、重量均提高；同一时期莲座叶叶片过表达植株叶片的衰老程度与野生型和突变体相比为更严重。对上述拟南芥不同株系进行生理生化分析,结果表明：当植株生长至50d时,突变体中的莲座叶叶片的叶绿素含量要显著高于野生型和过表达株系；在植株生长至60d、70d时,过表达株系中莲座叶片中的可溶性蛋白含量要显著高于野生型,而突变体与野生型的相比差异不明显；在40d以后,过表达株系中TBARS含量逐渐升高,在60d时株系中的TBARS含量显著高于野生型和突变体植株的含量；在植株生长至50d时,过表达株系中的POD、SOD、CAT酶活力都会明显下降,而脯氨酸含量在40d以后不断升高,与野生型和突变体植株的相比,差异极为显著。选取拟南芥中与叶片衰老相关的基因,采用荧光定量RT-PCR方法对拟南芥不同株系进一步分析了他们的表达情况,发现SAG12基因的转录水平在过表达株系中明显上调,而在突变体中明显下调；SAG113基因的表达在突变体中轻微下调,在过表达中轻微上调；NAP、NAC2基因的转录水平在过表达株系中上升了2倍左右,而突变体与野生型的相比差异不明显。另外,ATMYB2基因在突变体中的表达有所上调,但在过表达株系中其表达水平比野生型的高出了近5倍；WRKY6和WRKY53两个基因在突变体中的转录表达也轻微上调,但这二者的转录水平在过表达株系中都上调2倍左右。只有SAG29基因的表达在突变体和过表达株系中都呈上调的趋势。以上结果进一步表明：AT3G14067基因不仅直接与拟南芥的衰老相关,而且还可能通过调控其他衰老相关基因的表达促进拟南芥的衰老。选取拟南芥中与下胚轴发育调控相关基因,同样采用荧光定量RT-PCR方法,对拟南芥不同株系进行其转录表达量分析,发现ABCB19、SOB7和DFL1基因的表达水平在突变体中均显著上升,而三者在野生型和过表达株系中的表达水平差异均不显著；FHY3基因在突变体中有轻微上调趋势,而在野生型和过表达株系中的差异也不明显；而RLP21基因的表达水平在野生型和突变体中的差异不显著,在过表达株系中却呈现上调趋势；DWARF基因的表达在突变体中上调显著,但在过表达株系中却呈明显下调趋势；1EII、RAFL23、CUL1、RPT1、NPL1、HY3、LCL5、MAX2基因的表达在突变体和过表达株系中均呈现上调趋势,但在突变体中的上调程度都更高。选取与拟南芥花发育调控相关基因的荧光定量RT-PCR分析结果显示,AP2、ATlG01460基因在突变体中的表达水平有上调趋势,但在过表达株系中几乎与野生型的无差异；AP1、AG、AGL2和LFY3基因的表达水平在突变体和过表达株系中均显著下调；AGL9基因的表达在突变体和过表达株系中也均呈明显下调趋势,但突变体中的下调程度更显著；而FLC基因的表达水平在野生型和突变体中几乎无差异,但在过表达株系中的表达水平呈现下调趋势。以上结果表明：拟南芥AT3G14067基因还可能拟南芥的生长发育,如与下胚轴和开花调控等过程密切相关。更多还原

【Abstract】 Subtilisin-like proteases (subtilases) belong to the serine protease family(S8) which are widely distributed in all three kingdoms of life. This family of serine proteases is involved in multiple processes in plants, such as plants growth and development, response to environment changes and metabolic regulation. But their specific functions and regulation mechanism et al. are not entirely understanded. In the previous study of our group, a senescence-associated subtilisin-like serine protease was separated and purified from the dark-induced senescence wheat leaves, which was named TaSSPl (Triticum aestivum Subtilisin-like Serine Proteasel). The mass spectrometry data were used to alignment at NCBI protein database and only part of the protease amino acid sequence information was obtained for the database. However, the identified functions of this protease in wheat growth and development were not entirely clear. In this study, according to the known partial amino acid sequence of the protease, the RACE method was used to amplify the full sequence of TaSSP1protease gene. After the full-length sequence of TaSSPl gene was obtained, the CDS region of TaSSPl was used to compare in the MEROPS database and a protease of arabidopsis AT3G14067was found which with a homology was77.8percent compared to TaSSP1. The special function of AT3G14067protease was also not unclear in arabidopsis. Therefore, the methods of RT-PCR, real time quantitative PCR, western blotting, gradient gelatin-PAGE in gel proteolysis assay and transgenic et al. were used to furthermore understand the TaSSPl and AT3G14067function during plants development and response to environmental changes based on the results of the previous study. The main results obtained in this study were showed as follows:1) According to the known partial amino acid sequence of the TaSSP1, The RACE method was used to obtain the full-length sequence of wheat TaSSP1protease gene. The GenBank database accession No. was JX962746. This cDNA had a2361bp coding region, and was predicted to encode a protein of786amino acid residues, and that it contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI’s Conserved Domain Database (CDD).In order to gain a better understanding of the functions of TaSSPl, wheat were cultured and treated with100μM ABA,6-BA, ethephon, and200mM NaCl, D-Mannitol, sucrose or16%PEG6000respectively, whereafter the mRNA, protein and activity levels of TaSSP1in wheat at stated different hours after treatment were analyzed by RT-PCR, fluorescent quantitative RT-PCR, western blotting and gradient gelatin-PAGE in gel proteolysis assay. The results showed that the transcriptional and translational expression activities of TaSSP1in ABA treated wheat were gradually up-regulated with the extension of whole treatment time, but were obviously down-regulated in6-BA treated wheat from the middle to late stages of treatment. The increasing expression levels of TaSSPl were also identified in dark-induced senescence and older wheat leaves. In addition, the TaSSPl expression activities could be obviously up-regulated in wheats treated with200mM NaCl, D-Mannitol and sucrose or16%PEG6000. Furthermore, TaSSP1was showed to be a metal-dependent protease because its activity could be activated by some divalent metal cations, such as Cu2+, Ca2+, Zn2+and Mn2+. The results above indicated the function of TaSSPl was not only related with leaf senescence, but also related with the development of wheat under environment stress, especially to osmosis stress. And suggested that TaSSPl might be important on the response of wheat to environment stresses through its regulation of both gene expression and protein turnover.2) As AT3G14067protein sequence structure was analyzed through bioinformatics method, we found that AT3G14067has very similar structure compared to TaSSPl, and that it also contained a signal peptide, a19Inhibitor domain, a peptidase S8family domain, a protease-associated domain and a C-terminus domain as analyzed by NCBI’s Conserved Domain Database (CDD).Combined with semi-quantitative RT-PCR for analysis tissue-specific expression of AT3G14067, The result showed that the highest expression sites were in the stem, rosette leaves and cauline leaves which was consistent with the results of the plant membrane protein database. The expression of AT3G14067in response to various plant hormones,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000was further analyzed by Real-time quantitative PCR, the results show that it is apparently induced by ABA,6-BA, ETH,200mM NaCl,200mM D-Mannitol,200mM sucrose and16%PEG6000. These results indicated that AT3G14067might also be important in arabidopsis on the response to hormones and abiotic stress.In wild type arabidopsis, we also found that after the rosette leaves were treated with dark, the expression of AT3G14067was up-regulated after12hours treatment and came to the highest level at48hours treatment. In addition, The increasing expression levels of AT3G14067was also identified in older rosette leaves. This may indicated that AT3G14067gene was also a senescence-associated gene.In germination experiments of arabidopsis seeds, the results were showed that the expression level of AT3G14067was significantly up-regulated when seeds were cultured in dark, blue light and far-red light situations. Among them, the phenomena above in dark and far-red light treatments were most obviously. This indicated that the responses of AT3G14067gene were different in the seed germination treated with different light quality.The results preliminary showed that the AT3G14067protease was not only associated with arabidopsis leaf senescence, but may also closely related to the growth and development of Arabidopsis.3) Over-expression transgenic arabidopsis AT3G14067OX-lines3,4,6were obtained and identified as homozygous by real time quantitative PCR method. Compared with wild type, mutant and over-expression lines phenotype were found that when the seeds germinated in the dark condition, the hypocotyl of AT3G14067gene over-expression lines elongation significantly while the mutant and wild type plants had no significant difference between them. Moreover, the bolting time was5days in advance compared to the over-expression lines and the wild-type. However, the seeds of plants will mature early between the over-expression and the mutant plants. Under natural growth conditions, the plants height of the over-expression lines were lower. In contrast, AT3G14067mutants plants show the opposite phenotype. During the same periods, the rosette leaves of over-expressing plants had deeper leaf senescence degree compared to wild-type and mutant plants.The physiological and biochemical analysis showed that when the Arabidopsis thaliana grow to the50d stage, the content of chlorophyll in rosette leaves of mutant was significantly higher than that in wild-type and over-expression lines. At the60d and70d stage, the content of soluble protein in rosette leaves of over-expression lines was obvious higher than that in wild-type, while it in mutant is similar to wild-type plants. In over-expression lines, the TBARS content was beginning to increase after40d stage, and clearly higher than that in wild-type and mutant in the60d stage. In the50d stage, the enzyme activity of POD, SOD and CAT was significantly decrease, and the content of proline was rising significantly compared with the wild-type and mutant.The expression quantity of senescence-related genes in Arabidopsis thaliana leaves were analyzed by qRT-PCR. The expression levels of SAG12in over-expressing lines were significantly increased and in mutant significantly reduced, while SAG13in over-expressing lines were slightly increased and in mutant slightly reduced. NAP and NAC2in over-expressing lines were highly expressed, but in mutant and wild-type ware not significantly difference. The expression profiles for AtMYB2in over-expressing lines was5times higher than in wild-type. The expression patterns of WRKY6and WRKY53in mutant have a slight increase and in over-expressing lines have raised two times. Only SAG29appeared upward trend in both over-expressing lines and mutant. These results further indicate that AT3G14067gene not only directly related to aging, but also possibly promoting aging through regulation of other aging-related genes in Arabidopsis.Using the qRT-PCR method to analysis the genes associated with the hypocotyls development. The expression profiles for BCB19, SOB7and DFL1in the mutant were all significantly increased and in over-expressing lines and wild-type have slightly different. FHY3only in mutant has a slight upward trend. The expression level of RLP21in wild-type and mutant were similar while in over-expressing lines was up-regulated. The expression patterns of DWARF6in the mutant was significantly up-regulated but in over-expressing lines had an obvious downward trend. FE11, RAFL23, CUL1, RPT1, NPL1, HY3, LCL5, MAX2were up-regulated in both over-expressing lines and mutant, but in the mutant have a higher degree.The genes related to flowering were analyzed by the qRT-PCR method. Both AP2and AT1G01460in mutant had increased tendency, but had no change in over-expressing lines and in wild-type. The expression level of AP1, AG, AGL2and LFY3in mutant and over-expressing lines were significantly reduced. The expression profiles for AGL9in the mutant and over-expressing lines were all significantly up-regulated. The expression pattern of FLC in mutant and wild-type had no changed but in over-expressing lines had a downward trend.These results above suggest that Arabidopsis thaliana AT3G14067gene may also have close relationship to the plant growth and development, such as the development of hypocotyl and flowering in Arabidopsis thaliana..更多还原