1、沙冬青抗寒基因的克隆功能分析与遗传转化研究沙冬青抗寒基因的克隆、功能分析与遗传转化研究（作者：刘美芹，导师：尹伟伦教授）摘要 低温是影响植物正常生长发育的重要环境因子，揭露植物的抗寒机理，培育抗寒新品种具有重要的理论和实际意义。至今人们对植物低温抗性取得机制的了解仍很有限，尤其是对寒温带木本植物取得顺利越冬抗寒力的进程了解更少，而寒温带木本植物对维持北方酷寒地域的生态平衡起重要作用。本文针对生态建设中缺乏抗寒植物的生产急需问题，对我国北方内蒙古荒漠地域特有的常绿阔叶灌木沙冬青的抗寒性分子调控机制进行研究，期望为抗寒良种选育及生物工程定向育种提供理论和技术支持。沙冬青是亚洲中部的一种强抗逆植物，
2、生长在荒漠地带，是国家三级保护植物，对维持本地生态平衡起着重要的作用。沙冬青具有耐热、旱、盐碱等多种抗性，尤其能忍受冬季-30的低温，是理想的研究植物抗寒性的材料。目前对沙冬青抗逆性的研究多限于细胞组织结构的观察，虽然分离到几种热稳固的抗冻蛋白,但尚未从次生代谢物产物及抗逆基因方面进行研究的报导。据此，本文探讨了多酚与沙冬青抗寒性的关系；第一次用自行优化的固相扣除杂交方式，从低温驯化的沙冬青幼苗中克隆到多个低温诱导基因,并进行了功能分析与GenBank注册；将克隆到的其中一个基因AmCIP，通过烟草异位表达分析、亚细胞定位分析和原核表达分析等方式进行了功能鉴定和三倍体毛白杨的应用转化研究。主要
3、研究结果如下：针对沙冬青次生代谢产物含量很高的特点，就多酚类物质的含量与抗寒性的提高之间的关系进行了研究。将沙冬青幼苗在2-6条件下驯化14天后，检测发觉抗寒力明显提高。电镜细胞化学定位方式观察到经低温驯化的幼苗子叶细胞中，多酚类物质在液泡、液泡膜周围大量积累。定量分析发觉，低温驯化使沙冬青子叶内的多酚含量提高。说明低温驯化后，沙冬青中具有抗氧化剂活性的多酚的积累，可能对抗寒力的提高起重要作用。低温抗性是受多基因调控的数量性状。本文选用自行优化的固相扣除杂交技术，以分离克隆多个沙冬青低温抗性基因。该优化方式操作简单，快捷，集多种扣除杂交方式的长处于一体，能够同时分离取得多个全长基因。扣除后取得
4、的候选基因，经两轮杂交挑选后，取得11个不同显著且有编码区的克隆，别离在GenBank上进行了注册，序列号是 AY590122，AY843521，AY846361，AY846362，AY846363，AY846364，AY843522，AY843523，AY843524, AY843525，AY843526。这11个克隆中有三个是全长cDNA，含有完整的开放阅读框，其中两个编码区完全一致，都编码植物防御素；另一个编码植物LEA蛋白。其余的8个克隆中有两个是编码低分子量的低温-干旱诱导蛋白的基因，两个是编码光合系统元件蛋白的基因，一个H+转运ATPase的mRNA相似基因和一个14-3-3蛋白同
5、源基因。这11个克隆编码的蛋白属于不同的种类，可能参与渗透调节、光合系统调整、能量代谢及信号转导等，说明沙冬青的抗冻性形成进程至少有这些代谢进程的参与，揭露了沙冬青低温抗性的形成是受多因素调控的复杂进程。从以上挑选到的这些不同克隆能够看出沙冬青低温抗性取得进程的复杂性，但要了解其各个进程的作用机制，还需要对单个基因进行深切研究。因此，本文将低温驯化后表达量明显提高的克隆B12进行5端RACE扩增，取得全长806bp的类脱水素基因AmCIP，并进行了序列结构分析和功能预测。经基因组DNA扩增及序列分析发觉该基因没有内含子。Northern 杂交结果又表明AmCIP基因在对照材料中几乎没有表达，而
6、在低温驯化植株中却有大量表达，在驯化植株胚根中的表达量又明显高于子叶中的表达量。可见AmCIP的表达与沙冬青抗寒力的提高直接相关，尤其是能提高较幼嫩脆弱的胚根的抗寒力。为进一步鉴定AmCIP的功能，将AmCIP别离连接到35S启动子和rd29A启动子（Prd29A）下游，构建双元表达载体，并采用农杆菌介导的叶盘转化法转化烟草。转基因烟草的基因组PCR扩增, Southern及Northern杂交分析均证明目的基因已整合进烟草基因组中而且能在烟草细胞中表达。对T0代的多个转基因株系的低温抗性检测发觉低温下（-24）转基因烟草株系的离子渗漏率降低，膜稳固性增强，零下2处置后的存活率增加，尤其是转P
7、rd29A-AmCIP组合的株系。可见AmCIP的异位表达能明显提高植物的低温抗性，AmCIP可能具有低温保护蛋白功能，能在低温下保护细胞大分子及质膜的稳固性。由于AmCIP是类脱水素基因，推测能保护植物细胞抵抗水分胁迫。对叶片的保水力测定结果发觉，转基因烟草离体叶片的保水力比野生型烟草的保水力增强。PEG与NaCl处置进程中转基因烟草的生长状况优于野生型烟草，而且长时刻处置后存活的转基因烟草叶片都有发达的栅栏组织，细胞排列致密的海绵组织和排列整齐紧密的表皮细胞。由此可见，AmCIP的确能保护细胞免受脱水胁迫，协助维持细胞的正常生理功能。另外，为检测转基因烟草的遗传稳固性，将T1代转基因烟草的
8、种子在低温，PEG及NaCl胁迫条件下培育，发觉其萌生率及生长量均优于野生型烟草，暗示AmCIP基因的整合及表达能正常遗传。也进一步证明类脱水素AmCIP基因的异位表达能稳固提高转基因烟草对低温、干旱及盐碱等非生物胁迫的抗性。为进一步鉴定AmCIP的可能作用机制，将AmCIP和报告基因GFP融合构建了双元表达载体AmCIP:GFP并转化了烟草。在激光共聚焦显微镜下观察发觉转基因烟草的细胞质和细胞核中都有绿色荧光信号，尤其是细胞核中，荧光很强，说明AmCIP可能散布在细胞质、细胞核或结合在膜上（包括核膜），在低温下起保护膜及大分子的作用。为鉴定AmCIP的低温保护功能，构建了AmCIP原核表达载
9、体并转化大肠杆菌，诱导后目的蛋白能超量表达。对粗提菌液蛋白通过几丁质柱纯化，竟SDS-PAGE电泳取得了单一条带的21kD蛋白。将过柱纯化的蛋白煮沸十分钟后，电泳结果仍显示有单一条带的21kD蛋白；将纯化前的粗提蛋白煮沸后也一样能取得单一条带的21kD蛋白，说明AmCIP具有热稳固性。AmCIP在SDS-PAGE电泳迁移滞后的特性与其热稳固特性都是脱水素的特性，这进一步证明了AmCIP是类脱水素蛋白。纯化后的AmCIP还能提高低温条件下细菌的存活率，说明该蛋白对细胞具有低温保护功能。将构建好的目的基因上游是rd29A诱导型启动子的双元表达载体转化三倍体毛白杨。经基因组PCR鉴定，初步肯定目的基
10、因已整合进再生毛白杨植株中。荧光显微镜观察发觉转有融合AmCIP:GFP的再生植株的叶片和根中均有绿色荧光出现，说明Prd29A-AmCIP:GFP融合片断已整合进毛白杨的基因组中。总之，本文研究取得了多个沙冬青低温诱导基因，揭露了沙冬青低温抗性的提高是一个复杂的分子生理进程，是多种代谢途径多个基因协调作用的结果。主效基因AmCIP转化烟草异位表达表明AmCIP能提高植物对低温、干旱及高盐的抗性；亚细胞定位分析发觉AmCIP可能是在细胞质和细胞核中起保护作用；原核表达分析进一步验证了其保护功能。这些结果对深切探讨木本植物的抗寒性调控机制提供了理论依据；低温诱导基因的克隆、AmCIP的鉴定及转化
11、研究为林木花卉抗寒良种选育及生物工程定向育种提供了基因资源和技术支持。关键词：沙冬青，低温诱导基因，扣除杂交，遗传转化，烟草，三倍体毛白杨Cloning and characterization of cold-inducible genes of Ammopiptanthus mongolicus（Liu Meiqin Directed by Prof. Yin Weilun）Abstract Low temperature is one of major abiotic stresses that affect plant growth and distribution. Many spe
12、cies of plants are capable of adapting to low temperature and freezing conditions. Generally, this adaptation is not a constitutive but an induced process in response to low, but nonfreezing temperatures, a phenomenon known as cold acclimation. Although a number of genes have been correlated with th
13、e acclimation process, it is still unclear what are the roles of individual cold induced genes and how do they contribute to freezing tolerance development, especially in woody plants, even though their freezing tolerance constitute a key challenge in understanding the mechanism and forestry develop
14、ment in the future. In order to investigate multiple genes involved in cold acclimation of woody plants, an evergreen broadleaf species, A. mongolicus was adopted to dissect the cold acclimation. The rare endangered plant A. mongolicus is endemic to the Alashan desert, northwest sand area of China,
15、where the climate is arid, the site is desertification and salinization and the temperature is blow 30 in winter, up to 40 in summer. Situating in adverse environments, A. mongolicus has gradually evolved typical superxeromorphic structures and metabolic characteristics to adapt the adversity, such
16、as drought, salinization, cold, erosion and other stresses. Tolerance to various adversities makes A. mongolicus an ideal material for investigation of the mechanism of freezing tolerance for temperate zone woody plants. Thus far studies are mainly focused on its superxeromorphic structures, whereas
17、 investigations of the molecular changes in acclimation are comparatively scarce, besides the report that antifreeze proteins have been purified from the heat stable proteins in its winter leaves.It is known that A. mongolicus is abundant in secondary products, so the study was understaken to determ
18、ine the effects of cold acclimation on content and subcellular localization of phenolics in A. mongolicus cotyledon and the relation with freezing tolerance. A. mongolicus seedlings were acclimated at 6 day/2 night with 14 hours photoperiod (150 mol m-2s-1 photosynthetically active radiation) for 14
19、 days. Then cytochemical localization of phenolics in cotyledon cells was observed by electron microscopy using caffeine, which forms electron-dense deposits. Results showed that phenolics were mainly localized in the vacuole and in close proximity to tonoplast. Deposits also could be observed in th
20、e cytoplasm. No phenolic deposits were found in cell walls. After cold hardening, the amount of phenolics increased obviously in vacuole and in close proximity to tonoplast in cotyledon cells. Spectrophotometric measurement revealed that the content of phenolics in cotyledons of Ammopiptanthus mongo
21、licus grown at 25 was mg/gFw, whereas in the cold hardening seedlings it increased to gFw. As hydrogen- or electron-donating agents, phenolics protect plant cells against reactive oxygen species formed during chilling or freezing stress. So these results suggested that the amount of phenolics closel
22、y associated with the cold hardiness of Ammopiptanthus mongolicus seedlings.It is acknowledged that the ability of plants to cold acclimate is a quantitative trait involving the action of many genes with synergistic effects. So a modified solid-phase subtraction hybridization technique was developed
23、 to isolate and screen cDNAs whose transcripts increased in cold-acclimated A. mongolicus seedlings. The optimized strategy is simple, quick and full-length cDNAs can be obtained. After hybridization subtraction and two rounds of screen, differential cDNAs were selected and sequenced. Sequence analy
24、sis of the screened clones indicated that all of them hung with poly(A) and eleven clones had coding regions, with three of them containing complete open reading frame. And nine of the eleven clones shared various degrees of homology with the genes found in the GenBank database and the other two wer
25、e unidentified sequences. Sequence data further revealed that these accumulated transcripts encoded: three low molecular weight proteins a Late-embryogenesis-abundant (LEA) protein and two cold acclimation responsive proteins; two photosynthesis-related protein (photosystem I subunit II precursor (P
26、saD) and photosystem II oxygen-evolving complex 33kD subunit OEC33); a protease inhibitor; an adenosine triphosphatase and a 14-3-3 related protein. Analysis on the function of these proteins indicated that the low molecular weight proteins were associated with water holding ability of cytoplasm; ph
27、otosynthesis-related proteins participated in the adjustments of photosynthetic apparatus to resist photoinhibition; 14-3-3 related protein could interact with adenosine triphosphatase to enhance ATPase activity and energy metabolism; and protease inhibitor involved in the prevention of unwanted cel
28、l death caused by reactive oxygen species. We suggest that cold acclimation with low light intensity in A. mongolicus is a more complex interaction of low temperature, light, energy and signal than that assumed previously.The clone with most strong signal among all the screened clones was selected f
29、or continuing research. Its 5 sequence was obtained by 5 RACE and then full-length cDNA was cloned based on the 3sequence and 5 sequence. The full-length cDNA designated as AmCIP (Ammopiptanthus mongolicu cold-induced protein) gene was 806bp long and contained a 465bp open reading frame (ORF) encodi
30、ng a kD protein of 154 amino acids. Bioinformatic analyses indicated that CIP belonged to dehydrin family with the features of high hydrophilicity, a helix K-segment, a long Gly-rich region and a phosphorylatable tract of Ser as well as deficiency in Cys and Trp. The expression of CIP gene increased
31、 after two weeks of cold treatment and more expression was detected in radicle than in cotyledon. And the result of PCR amplification the CIP gene from genome of A. mongolicus revealed this gene has no introns. Function prediction suggested this protein may protect the stabilization of membrane stru
32、cture and prevent macromolecualr coagulation or sequestrate calcium ions under low temperature conditions.The function of AmCIP was characterized by ectopic expression and abiotic stresses tolerance assay of transgenic tobaccos. Full length AmCIP was ligated downstream of 35S promoter and rd29A prom
33、oter cloned from Arabidopsis and inserted into binary expression vector pBI121 to construct new vectors S-cip and R-cip. Then these vectors were transformed into tobaccos, respectively. Genomic DNA PCR amplification, Southern blot and Northern blot showed the target fusion gene fragment had integrated into the genome of tobacc
