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作者	单位
陈冬阳，杨明明，高翔，张龙龙，郭江岸，李万	（1.西北农林科技大学农学院，陕西杨凌 712100； 2.陕西省小麦新品种培育工程研究中心，陕西杨凌 712100）

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中文摘要:

几丁质酶是植物重要的防卫因子之一，与植物抗病性密切相关。为深入了解几丁质酶基因表达及功能，先利用引物ChiF/ChiR从10份具有不同抗病性的小麦中克隆出几丁质酶基因后对其进行序列分析及原核表达分析，再利用荧光定量PCR（qRT-PCR）技术分析小麦根、茎和叶不同组织中几丁质酶基因的表达情况，并选取在小麦根、茎和叶中表达量最高的几丁质酶基因进行真菌性病害的抗性反应试验。结果表明，从10份具有不同抗病性的小麦中克隆得到5条长度约960 bp的几丁质酶基因序列，其中1条序列与已公布的 Chi-3基因序列（序列登录号为KJ507387）一致，其余序列命名为 Cht1~Cht4(GenBank登录号为KR049247～KR049250)。序列分析表明，克隆所得序列无内含子，编码的几丁质酶属于ClassⅠb类型，也属于糖苷水解酶第19家族，是胞外分泌蛋白且兼具溶菌酶活性。SDS-PAGE分析表明，重组质粒pE-Chi能在BL21(DE3)中表达一个33 kDa左右的特异蛋白。qRT-PCR分析表明，这5种几丁质酶基因在小麦根、茎和叶中均有表达，但在不同器官中表达量差异较大，其中，叶中表达量最高，根中次之，茎中最低；同时，这5种几丁质酶基因在同一器官中表达量大小具体表现为 Cht4> Cht2> Cht3> Cht1> Chi-3。选取相对表达量最高的 Cht4基因验证其对真菌性病害的抗性反应，结果表明，在条锈菌胁迫下， Cht4基因在抗病材料92R137中的表达量显著高于感病材料铭贤169，并且均高于同等条件下接ddH₂O的表达量。本研究扩增到的几丁质酶基因在小麦中组成型表达，说明其参与了小麦的正常生长发育过程； Cht4基因的表达受条锈菌诱导，推测其可能参与了小麦叶部抗条锈菌的防御反应。

英文摘要:

Chitinase,an important defensive factor in plants,is closely related to plant disease resistance. In order to understand the expression and function of chitinase genes,by the using of primer ChiF/ChiR,chitinase genes were cloned by PCR from 10 wheat cultivars with different resistance to disease in this study. After that,these sequences were analyzed by different biology software,and were expressed in E.coli.The expression of these genes in root,stem and leaf were analyzed by quantitative real-time PCR. The highest expression of chitinase gene in these tissues was selected to test the resistance with fungal diseases. The result showed that 5 chitinase gene sequences (960±3 bp in length) were cloned from 10 wheat cultivars with different resistance to disease. Of them,one sequence is consistent with the published Chi-3 gene sequence with the accession number KJ507387,and the others were named Cht1 to Cht4 with the accession numbers of KR049247 to KR049250,respectively.Sequence analysis showed that the chitinase genes in this research have no intron,encoding chitinase belonging to ClassⅠb type. The chitinase are the members of glycoside hydrolase family 19,which are extracellular secreted protein and have lysozyme activity. The results of SDS-PAGE showed that the recombinant plasmid pE-Chi could express a 33 kDa protein after induction in BL21(DE3).The results of quantitative real-time PCR showed that these five kinds of chitinase genes were expressed in wheat roots,stems and leaves. However,the amount of expression in different organs are quite different. The content of each chitinase gene in this experiment is highest in leaf,followed by root,and is minimum in stem. Meanwhile,the expression of these five kinds of chitinase genes in the same tissue is ranked as: Cht4> Cht2> Cht3> Cht1> Chi-3. Therefore, Cht4 gene,which has the maximum content in the same organ,was selected to validate its resistance to fungal diseases. The results showed that under the stripe rust stress,the expression of Cht4 gene in 92R137 (resistant material) was significantly higher than in Mingxian 169 (susceptible material),and both of them were higher than control. In this study,the chitinase genes are constitutively expressed in wheat,indicating their participation in the regular growth and development of wheat. The expression of Cht4 gene is induced by stripe rust,suggesting that it may be involved in the defense reaction for stripe rust in wheat leaves.

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