【作者】 何涛；

【导师】 贾敬芬；

【作者基本信息】 西北大学 ， 植物学， 2007， 博士

【摘要】 温度是限制植物地域分布的一个主要因素，大多数植物在低温下一般会遭到不同程度的伤害，全球每年由于低温冻害造成的作物损失巨大。因此，研究并提高植物的抗寒性具有重要的理论和现实意义。近年来，由于生物技术的发展和广泛应用，大量与抗寒相关的基因被分离和鉴定出来，并证明其中部分基因对提高植物的抗寒性有一定作用。同时，初步提出了低温信号转导以及调控路径的模型，从而为采用基因工程手段进行植物抗寒育种及有关生理研究提供了新的启示。青稞，又称裸大麦，是一种禾本科作物，主要分布于青藏高原地区。与其他禾本科作物相比，青稞对极端环境胁迫，尤其是对低温胁迫的抵抗能力较强，是一个潜在的遗传资源库，研究并开发这些抗低温相关基因及其调控机制将具有十分重要的理论和实践意义。为此，本文以青稞作为实验材料，在建立起其组织培养离体再生体系的基础上，对其冷诱导相关基因进行了研究，结果如下：Ⅰ．以青稞成熟胚为外植体，对外植体作两种处理：(1)成熟胚外带部分胚乳(E)；(2)成熟胚不带胚乳(NE)。两种成熟胚均接种在含不同浓度2，4-D(1～5 mg／L)的MS培养基上。暗培养7 d后，白色疏松的愈伤组织出现，同时，在外植体E中部分胚直接长出芽，胚的发芽率随2，4-D浓度的升高呈下降趋势。在两种外植体中，愈伤组织诱导率存在一定差异。外植体NE产生的愈伤组织明显高于外植体E，在2，4-D浓度为3 mg／L时，其诱导率可达93.1％。愈伤组织在含2 mg／L 2，4-D的激素条件下继代培养1代后转入含不同浓度6-BA(1～5mg／L)和500 mg／L CH的MS培养基上，21 d后愈伤组织表面开始出现绿色芽点。与愈伤组织诱导相反，来源于外植体E的愈伤组织的分化率高于外植体NE的愈伤组织，在含2 mg／L 6-BA的培养基上，计有65.2％的愈伤组织可分化出苗。转入1／2 MS培养基上则可诱导出根。研究建立了青稞成熟胚离体诱导再生植株的实验体系。Ⅱ．采用PCR法从青稞中克隆了hblt14.2基因，与大麦的blt14.2基因具有98.9％的同源性，在蛋白质上存在2个氨基酸的差别。hblt14.2基因共有470个核苷酸，其中包括249 bp的开放阅读框，69 bp的5’非翻译区和152 bp的3’非翻译区。该基因编码一个82个氨基酸的蛋白质，分子量为7.71 kDa，等电点为7.03。氨基酸组成表明，该蛋白富含Gly(17.07％)、Ala(15.85％)、Leu(13.41％)和Val(9.76％)氨基酸，是一种高度亲水的小分子蛋白。hblt14.2蛋白二级结构主要以丰富的α-螺旋和无规则卷曲为主，与其他冷诱导基因在氨基酸组成和二级结构上具有相似性。采用RT-PCR方法研究了青稞hblt14.2基因在低温、盐胁迫以及ABA处理下的转录水平变化。结果表明，只在4℃低温处理下的幼苗中检测到了hblt14.2基因的表达，而在盐胁迫和ABA处理的幼苗中未检测到，推测hblt14.2基因为一个专一低温诱导基因。此外，RT-PCR分析显示，hblt14.2基因的表达可能具有组织特异性，它在叶片中的表达量很高，但在根中未检测到该基因的表达。将hblt14.2基因连上强启动子CaMV35S和终止子后定向插入质粒pCAMBIA1301的多克隆位点中，构建了植物表达载体pCAMBIA-BI-hblt。通过根癌农杆菌LBA4404介导，将连有启动子和终止子的目的基因转入普通烟草。PCR和Southern检测表明，目的基因已整合到烟草基因组中。RT-PCR检测表明，目的基因在转基因烟草中获得了转录水平上的表达。采用酶法分离了转基因烟草及其对照植株的叶肉原生质体，在相同条件下作低温胁迫培养。结果表明，转基因烟草原生质体存活率略高于对照植株，低温胁迫35 d后，转基因植株原生质体存活率为56.7％，而对照植株为43.2％。低温处理1 w后，在转基因植株的原生质体中出现了1次细胞分裂，而在对照植株中未出现。此外，对低温胁迫下转基因烟草及其对照植株的叶片脯氨酸含量进行了测定。结果表明，转基因烟草中的脯氨酸含量低于对照植株。在低温处理72 h后，对照叶片中脯氨酸含量为1.22 mg／g，而转基因烟草中只有0.57 mg／g。分析认为，可能是hblt14.2基因在烟草中的高效表达，在一定程度上提高了植株对低温的抵抗力，从而使另一条抗低温代谢途径——脯氨酸合成途径被削弱。由上述结果推测，青稞hblt14.2基因与植物的抗寒性有一定关系。Ⅲ．以4℃低温处理的幼苗为实验方(Tester)，室温生长的幼苗为驱动方(Driver)，通过抑制差减杂交(SSH)技术构建了包含240个单克隆的青稞差减cDNA文库。从文库中随机取出32个克隆进行双酶切检测，结果表明87.5％的克隆中含有插入片段。对其中16个克隆进行测序，获得12个不重复cDNA序列，片段平均大小为452 bp。序列比对显示，这些片段与其他植物的下列基因具有一定的同源性：金属硫蛋白、蛋白激酶、乙烯信号转录因子、bzip转录因子、锌指转录因子、1，5-二磷酸核酮糖羧化酶、富含亮氨酸蛋白、果糖二磷酸酶、铜锌超氧化物岐化酶、核糖体蛋白、钠氢逆向转运蛋白、过氧化氢酶、NADPH-细胞色素还原酶、抗坏血酸过氧化物酶、DNA结合蛋白和糖转运蛋白等。分析表明，本文构建的差减文库的确包含的是和低温诱导相关的一些cDNA克隆。同时，序列比对也暗示，青稞对低温的适应是通过多种途径来实现的，是多基因共同作用的结果。青稞差减cDNA文库的构建为进一步分离克隆低温诱导相关基因建立了前期工作。更多还原

【Abstract】 Temperature was one of important factors influencing plant growth and distribution. Most of plants would be suffered from chilling or freezing injury under low-temperature stress. Every year, the loss led by cold in crops was very heavy in the world. Therefore, it was theoretically and practically significant to study and enhance the cold-tolerance in plants.With the development and wide application of biotechnology, some of cold-tolerant genes were isolated and identified from plants in recent years, and the evidences showed that a part of them played important roles in resisting cold stress in plants. Moreover, some hypotheses and models about low-temperature signal transduction and regulatory mechanisms were put forward. These works provided a new way for cultivating cold-resistant species by genetic engineering technique.Highland barley (Hordeum vulgare L. var. nudum HK. f.), belonging to the genus Hordeum in the family Gramineae, distributed mainly in the Qinghai-Tibet Plateau, China. It has stronger cold-tolerance than other cereals, and was a potential resource of cold-resistant genes. Therefore, highland barley was used as materials in this study, the cold-induced genes cloned from it were studied on the basis of established high efficient regeneration system. The results obtained were as follows:I. In vitro protocol for efficient plant regeneration has been established from mature embryo-derived calli of highland barley. Embryos with part of (endosperm; E) or without endosperm (non-endosperm; NE) were excised from mature seeds and cultured on MS medium supplemented with 2, 4-D (1～5 mg/L) for callus induction. Percentage of callus induction from E explar ts was significantly lower than that from NE. The highest frequency (93.1%) of callus induction was obtained from NE explants on MS medium containing 3 mg/L 2, 4-D. The viable calli were subcultured at reduced concentration of 2, 4-D (2 mg/L), and then transferred to MS medium supplemented with 6-BA (1～5 mg/L) and 500 mg/L CH for shoot regeneration. Contrarily, percentage of plant regeneration from E explant-derived calli was higher than that from NE. The highest frequency of plant regeneration (65.2%) was obtained from E explant-derived calli on MS medium containing 2 mg/L 6-BA. The regenerated shoots were multiplied and rooted on half-strength MS medium without growth regulators. The rooted shoots were transplanted to soil with 100% survival. This method could be employed for the genetic manipulation studies.II. A cold-induced gene was cloned from highland barley by PCR, named hblt14.2, which exhibited 98.9% homology to bltl4.2 of barley, and there were 2 amino acids difference between hblt14.2 and blt14.2 proteins. The length of hblt14.2 was 470 bp, containing a 249 bp ORF, 69 bp 5’ untranslated region (UTR) and 152 bp 3’ UTR. Hblt14.2 gene encoded a protein of 82 amino acids with a calculated molecular weight of 7.71 kDa and a isoelectric point of 7.03. Hblt14.2 protein was a highly hydrophilic small protein with rich Gly(17.07%), Ala(15.85%), Leu(13.41%) and Val(9.76%) amino acids. The secondary structure analysis showed that hblt14.2 protein was rich in alpha helix and random coil, which were similar to other cold-induced genes.Hblt14.2 expression patterns under cold and other abiotic stresses were analyzed by RT-PCR. The results showed that the expression of the gene could only be induced by low temperature, while there were not expression under salt stress and ABA treatment. Thus, it was speculated that hblt14.2 was possibly a low temperature-specific gene. Furthermore, the level of hblt14.2 expression was higher in the leaves than in roots when plants were exposed to 4℃, which suggested that hblt14.2 expression had probably tissue specificity.Hblt14.2 gene linked with CaMV 35S promoter and terminator were inserted into multiple clone site in plasmid CAMBIA1301, which constructed the plant expression vector pCAMBIA-BI-hblt. Then, the target gene was transformed into Nicotiana tabacum by Agrobacterium tumefacien LBA4404 as vector. PCR and Southern examination showed that hblt14.2 had been inserted into tobacco genome. Hblt14.2 expression pattern in tobacco under cold stress was analyzed by RT-PCR. The results indicated that the gene could be transcribed correctly in transgenic tobacco.The mesophyll protoplasts were isolated through enzyme digestion from transgenic tobacco plants and the controls. Subsequently, they were cultivated under 4℃stress. The results showed that the survival rates of protoplasts from transgenic plants were higher slightly than that of the controls. After 35 days under cold stress, the survival rate of protoplasts from transgenic plants was 56.7%, whereas the controls was 43.2%. First cell division was observed in transgenic tobacco protoplasts after 1 week of culture, but no division was observed in the controls. Moreover, the contents of proline were analyzed in leaves of transgenic tobaccos and the controls under 4℃stress. The results showed that the proline contents were lower in transgenic plants than that in the controls. After 72 hours under cold stress, the proline content was 1.22 mg/g in the controls, while in transgenic plants was 0.57 mg/g. It was speculated that the cold-tolerance of tobaccos was possibly enhanced partly owing to hblt14.2 gene overexpression. As a result, other cold-resistant pathways such as proline synthesis pathway might be weakened. The results as stated above suggested that hblt14.2 was related to cold resistance in plant.III. Cold-induced genes of highland barley were studied by using suppression subtractive hybridization (SSH) technique, in which the cDNA from the materials treated with 4℃was used as "tester" and the cDNA from the materials growing in green house as "driver". A subtractive library of highland barley including 240 cDNA clones was constructed in the study. Enzyme digestion of 32 clones chosen randomly from the library indicated that there were inserts in 87.5% of them. The cDNA inserts of 16 clones were sequenced and blast searches were performed. Blast analysis showed that these cDNA were homology to the following genes: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1, 5-bisphosphate carboxylase, leucine rich protein, fructose-bisphosphate aldolase, copper/zinc superoxide dismutase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein and sugar transporter-like protein. The results indicated that the cDNA clones in the library were related to cold-induced genes. It was suggested that the cold-tolerait mechanism of highland barley might be a complicated, interactive system involving multiple approaches and multiple genes. Construction of subtractive cDNA library provided a advantage for further studies to isolate and clone cold-induced genes in higriland barley.更多还原