【作者】 郑佳秋；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2009， 硕士

【摘要】 遇到高温胁迫时植物体内会产生应激蛋白——热激蛋白,其中植物小分子量热激蛋白含量最为丰富、种类繁多。小分子量热激蛋白的分子量在17-30 kDa之间。高等植物中含有至少6种小分子量热激蛋白,分别定位于细胞质、叶绿体、线粒体、内质网。在常温下植物体内小分子量热激蛋白基因不表达,高温时小分子量热激蛋白则快速积累。近年来,关于高温诱导热激蛋白以及热激蛋白提高植物耐热性的研究取得了重要进展。然而,关于高温诱导热激蛋白基因及其在低温防御中作用的研究相对较少。本研究从不结球白菜中分离到小分子量热激蛋白基因BcHSP,表达模式和功能分析表明,这个基因的表达受高温逆境诱导,高温、低温条件下过量表达对大肠杆菌均有保护作用,构建了过量表达载体进行了转基因初步鉴定,为进一步BcHSP基因研究打下基础。1.利用同源序列设计兼并引物,通过RT-PCR和RACE方法从不结球白菜叶片中克隆到小分子量热激蛋白基因的中间片段,通过5’-RACE和3’-RACE分别克隆到5’片段和3’片段,拼接后设计特异引物扩增到全长722bp cDNA,命名为BcHSP(DDBJ登录号：AB367955),ORF为473 bp,编码157个氨基酸的多肽,理论等电点(pI)为5.31,分子量约为15.98 kDa。核苷酸序列同源性比较发现,BcHSP与芥蓝相似性最高达98%。氨基酸序列进行比对发现,相似性均在65%以上,说明HSP具有高度保守性。HSP的高度保守性说明他们在生物生命活动中具有重要的作用。聚类分析结果显示,BcHSP基因与芥蓝(CAB93512)最先聚为一类,亲缘关系最近,芜菁(AAB72190)单个聚一类、然后与拟南芥(NP-175759)、豌豆(P19243)、大豆(P02519)等聚为一类,最后与水稻(EAY89381)聚类,亲缘关系相对较远。实时定量检测表明,不结球白菜BcHSP转录表达受热激诱导,以叶片中表达量最高,BcHSP在不结球白菜叶片中表达特征说明它可能与植物叶片的耐热性关系更为密切。2.不结球白菜幼苗经42℃热激后,其幼苗的耐冷性明显增强,膜伤害程度降低。热激处理组的冷害指数仍低于对照组,差异显著(P<0.05),并且经热激的幼苗鲜重比对照增加了51%,苗高增加了40%。热激使不结球白菜幼苗在冷胁迫和恢复生长过程中电解质的渗透和丙二醛(MDA)含量均比对照大为下降,差异显著(P<0.05),显示热激降低了不结球白菜幼苗的脂质过氧化水平,并且使幼苗的抗氧化系统中的关键酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性在冷胁迫及恢复生长2d时仍保持比对照组高,差异显著(P<0.05)。热激处理后与对照组的幼苗SOD同工酶的电泳图谱显示,热激诱导使幼苗在低温胁迫前后SOD同工酶谱带均比对照组强,以上与冷害相关的生理生化指标测定结果显示,热激处理增强了不结球白菜幼苗的耐冷能力。荧光实时定量分析表明BcHSP的表达量与耐冷性相一致。低温(4℃)胁迫下,在大肠杆菌中异源表达BcHSP可维持大肠杆菌较高的细胞活力,这些结果表明BcHSP合成与不结球白菜幼苗耐冷性的提高有关。3.本实验从不结球白菜中克隆BcHSP基因的cDNA完整编码区序列,用来构建植物过量表达载体,并将表达载体转化到农杆菌菌株LBA4404中,以根癌农杆菌介导法转化不结球白菜‘苏州青’,GUS组织化学染色,发现大多数材料均出现了蓝色斑点,呈阳性,说明农杆菌的侵染效率较高,初步验证植物表达载体已经整合到植物组织中。PCR检测结果表明：目的基因已经整合到苏州青基因组中,为深入研究BcHSP基因功能创造了条件。更多还原

【Abstract】 All organisms respond to elevated temperatures with production of a defined set of proteins called heat shock protein. The small heat shock proteins (sHSP) are ubiquitous in nature.The sHSPs range in size from 17 to 30 kDa. In higher plants six nuclear gene families encoding sHSPs have been defined. Each gene family encodes proteins founding a distinct cellular compartment, including the cytoplasm,chloroplast, mitochondrion and ER. In general, the sHSPs are not found in normal vegetative tissues, but accumulate to high levels in response to heat stress.Recently, a salient process involved in the production of sHSP induced by heat shock and the relationship between sHSP and thermotolerance was proposed.However,little is known about the production of sHSP induced by heat shock and the relationship between sHSP and chilling tolerance.In this study, we cloned the characterization of BcHSP genes from non-heading Chinese cabbage.The expression pattern and functional analysis showed that expression of the genes was induced by heat stress, and sHSP play a role in protection of the proteins in bacteria,under low temperature and high temperature.In this experiment, a plant over-expression vector was constructed, which provided an effective tool for the further study of BcHSP gene function.1. Degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from non-heading Chinese cabbage on the homologous sequences from other plants.The middle fragment of interested cDNA was obtained by RT-PCR.The full length cDNA of small heat shock protein (sHSP) was isolated by 5’-RACE and 3’-RACE. The gene was designated as BcHSP (DDBJ accession number AB367955),which contained 722bp nucleotide and encoded 157 amino acids, PI was 5.31,molecular weight was about 15.98kDa.Further comparison showed its similary was above 98% with Brassica oleracea. amino acid comparison showed its similary was above 65%,which indicated HSP was high conservation,which indicated HSP play a important role in life activity.The phylogenetic analysis revealed genetic relationship of BcHSP and Brassica rapa was nearest,then was A-rabidopsis thaliana,Pisum sativum,Glycine max, the fartest was oryza sativa.Real-time qu-antitative PCR analysis revealed the BcHSP gene was obviously expressed with heat-shock ,which was accumulated most abundantly in non-heading Chinese cabbage leaves. Expression characterization of BcHSP in non-heading Chinese cabbage leaves indicated that it might be more correlative with increase heat tolerance of leaves than other tissues.2. Heat shock(42℃) applied to non-heading Chinese cabbage seedling increased the chilling tolerance of seedling and decreased the permeability of cellular membranes.Chilli-ng injury index was higher than control,difference was significant(P<0.05).Fresh weight and seeding height was higher than control 51%,40% respectively. The leakage of electrol-yte and malondialdehyde content were depressed,in the period of Chilling stress and recovery,difference was significant(P<0.05),indicated heat shock decreased lipid peroxide-ation level.In contrast, The activities of superoxide dismutase, catalase, and peroxidase were increased,difference wassignificant(P<0.05).The map of SOD isozyme showed the band of SOD isozyme is stronger than control,these results indicated heat shock increased the chilling tolerance of seedling.Real-time quantitative PCR analysis revealed that the BcHSP gene expression was coincide with chilling tolerance of seedling. The heterologous expression of BcHSP could maintain higher cell viability of E.coli under low temperature stress(4℃). These results indicated that the BcHSP synthesis was correlated with the increased chilling tolerance of non-heading Chinese cabbage seedling.3. In this experiment, a plant over-expression vector was constructed, which consists of the sence of BcHSP cDNA sequence from non-heading Chinese cabbage. Vecotor transfo-rmed into agrobacterium LBA4404,transformation "Suzhouqing" by a grobacerium tumefaciens.GUS chemical stain showed many materials expression blue band,which indicated colonization rate was high and preliminary identification vector had transformed into plant tissue.PCR analysis revealed that BcHSP gene had integrated to non-heading Chinese cabbage (Suzhouqing) genome, which provided an effective foundation for the further study of BcHSPgene function.更多还原