CCM: Cloning and expression of chitinase genes in wheat 07-05-2016

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 research.



                                                                                 Source: Baidu


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 Classb 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 amounts 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 the 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 research, 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.


This article comes from Seed China News 1606, CCM



 

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Tag: wheat


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