摘要
深入挖掘苜蓿属牧草的抗寒基因资源，揭示其抗寒机理，为苜蓿属牧草的抗寒育种研究提供理论依据。以该属抗寒性较强的直立型扁蓿豆为材料，低温冷诱导3-5叶期幼苗，利用cDNA-AFLP和RT-PCR同源序列克隆技术，筛选克隆扁蓿豆抗寒相关基因，进一步通过荧光定量PCR技术和生理生化指标测定，分析扁蓿豆抗寒相关基因与其他植物抗寒基因的差异、表达时期及基因表达与生理生化指标间的相关性等表达特性，为明确其抗寒机理奠定基础。主要研究结果如下：
1．直立型扁蓿豆幼苗的叶、茎、根对低温有不同程度的响应，可溶性糖含量和POD活性最先在根中提高，最高水平量是对照可溶性糖和POD活性量的3.64和1.64倍；可溶性蛋白和SOD活性最先在叶中提高，最高水平量是对照可溶性蛋白和SOD活性量的1.52和2.12倍。可溶性蛋白、SOD酶活性和POD酶活性间存在显著相关性，说明扁蓿豆在低温胁迫条件下提高渗透物质含量和抗氧化物酶活性来抵御寒冷。
2．通过RT-PCR技术同源克隆得到扁蓿豆Actin、CAS15A和CAS15B基因，与苜蓿的Actin和CAS基因相似率高达93%以上。其中扁蓿豆Actin基因在不同温度处理下的叶、茎、根中表达均无差异，可作为后期定量分析的内参基因；扁蓿豆CAS基因的核苷酸序列含有CRT/DRE（TACCGACCA）低温应答元件，同时氨基酸序列具有与其他植物CAS基因相似的4个相关位点、4次重复的10肽序列的低温相关位点和序列；扁蓿豆CAS15基因属于CAS冷诱导基因家族，随着低温胁迫时间的延长表达量呈现上升趋势，并在冷处理72h和84h达到相关表达量最高水平，推测CAS15的表达增强了扁蓿豆的抗寒性。
3．采用EcoRⅠ和MseⅠ为限制性内切酶建立扁蓿豆的cDNA-AFLP反应体系，用于扁蓿豆低温诱导基因差异表达的研究。选取其中58条上调表达的差异片段进行功能预测，主要参与光合作用、呼吸反应、细胞信号转导、物质代谢、防御反应等生理过程，其中70.79%的差异片段序列与蒺藜苜蓿的相关序列高度一致。通过qRT-PCR对其中7个有特点的基因进行量化分析证实均参与低温调控。
4．利用RACE技术获得扁蓿豆抗寒相关的MRPP2C、MRHSP70、MRHLH的cDNA，其编码蛋白分别具有PP2C、HSP70、HLH的保守域，与蒺藜苜蓿及其他植物的相应基因编码氨基酸序列具有很高相似度。在低温处理84h内，3个基因在扁蓿豆幼苗中的表达量变化趋势相似，均出现了2个峰值。其中MRHLH与MRCAS15基因表达量呈显著正相关，SOD酶活性也与抗寒相关基因MRHLH、MRCAS15A、MRCAS15B呈显著正相关。通过分子水平和生理水平抗寒研究，说明扁蓿豆抗寒能力是一个复杂的调控网络。
Digging deep into cold resistance genes of Medicago and revealing the mechanismcan provide theoretical basis for cold resistance breeding on Medicago. In this paper,Medicago ruthenica which has strong cold resistance were taken as materials.cDNA-AFLP and RT-PCR were employed to analyze and to isolate cold stress genesexpressed under cold stress using the three-five leaf stage leaves of Medicago ruthenica.The results of qRT-PCR and physiological characteristics were used to analysize thedifference between cold resistance gene of Medicago ruthenica and other plants’ relatedgene and express stage, the correlation on gene express and physiology and biochemistryindexes. The studies can lay the foundation for getting results clearly on cold resistancemechanism. The main results were as follows:
1. The seedling leaf, stem and root of vertical Medicago ruthenica were varyingdegrees of response to low temperature. Soluble sugar content and POD activity firstincreased in the root, the highest level was3.64and1.64times as the control; solubleprotein content and SOD activity first increased in leaves, the highest level was1.52and2.12times as the control. Soluble protein content and SOD activity had significant positivecorrelation with POD activity. The results showed that Medicago ruthenica could againstthe cold by increasing the content of osmotic substances and the activity of anti-oxidativeenzyme under low temperature.
2. The mRNA of Actin, CAS15A and CAS15B on Medicago ruthenica were cloned byRT-PCR, and the blast analysis showed that the above genes had93%nucleotide similarityto the Actin and CAS gene of alfalfa. The MRActin gene expression had no significantdifferences in leaves, stems and roots under the different temperature treatments, whichcan be as a reference gene for the quantitative analysis. The MRCAS gene harboredCRT/DRE(TACCGACCA）regulatory motifs, four dehydrin segments and a decapeptidemotif repeated four times similarity with the CAS genes in other plants. MRCAS15genebelonged to the CAS family. Real time PCR ananlysis revealed that the expressions ofMRCAS15A and MRCAS15B were steadily up-regulated under low temperature stress, andthe highest expression level at72h and84h, then the result declined the expression ofCAS15gene could enhance the cold resistance of Medicago ruthenica.
3. The cDNA amplified fragment length polymorphism (cDNA-AFLP) analysissystem was established with the restriction endonuclease EcoRⅠand MseⅠ to analyzegenes differentially expressed under cold stress of Medicago ruthenica.58up-regulatedTDFs involved in photosynthesis, respiration, cell signal transduction, metabolism, defensereaction and many other important process. Blastn and blastx analysis showed that morethan70.79%of the up-regulated TDFs had identity genes with Medicago truncatula. Theresults of quantitative analysis showed that7characteristic genes were involved intemperature regulation.
4. The cDNA of MRPP2C, MRHSP70and MRHLH were induced by RACE.Sequence analysis showed that the amino acid residues included conserved domains PP2C,HSP70and HLH, which had high homology with corresponding protein of Medicagotruncatula and many other plants. The qRT-PCR results indicated that the expression levelsof three genes shared sililar tendency and had two high peaks within84h of lowtemperature stress treatments. The expression of MRHLH gene had significant positivecorrelation with MRCAS15gene. The cold-resistant genes MRHLH, MRCAS15A andMRCAS15B had significant positive correlation with SOD activity. The cold ability was acomplex regulatory network of Medicago ruthenica from molecular level and physicallevel.
引文
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