芥菜新型几丁质酶基因在番茄中表达及其功能研究

The novel plant chitinase gene BjCHI1 was cloned from Brassica juncea in 1999. It encodes a chitinase, BjCHI1, with two chitin-binding domains that is different from all the chitnase genes isolated previously. In order to investigate the functions of the two chitin-binding domains of the BjCHI1, Zhao et al (1999) tried to express BjCHI1 in Escherichia coli expression system which failed. Leung et al (2002) expressed BjCHI1 in transgenic tobacco plants but it is very hard to get enough purified BjCHI1 for functional analysis. In this study, we have successfully expressed BjCHI1 in Pichia pastoris expression system in which the expressed target protein secreted into the liguid medium and large amount of target proteins can be obtained for functional assays. We have shown that the novel protein BjCHI1 displays both chitinase and agglutination activity. .Baced on the sequence of BjCHI1 cDNA, three special primers were designed and used in polymerase chain reaction (PCR) to amplify the fragments of BjCHI1 encoding the mature protein with two chitin-binding domains, BjCHI2 encoding the protein with only one chitin-binding domain and BjCHI3 encoding the protein without chitin-binding domain. The amplified fragments were cloned into pPIC9k vector, generating yeast expression plasmids pP17, pP28 and pCat, respectively. Then the three constructs were transferred into yeast KM71 and strains that could highly express the genes by secreting the proteins BjCHI1, BjCHI2 and BjCHI3 were selected, respectively. Western blotting analisis was carried out to confirm the target proteins. The chitinase activity of Fast Performance Liquid chromatography (FPLC) purified BjCHI1, BjCHI2 and BjCHI3 were tested and the results shown that all the three proteins degraded both CM-chitin-RBV and colloidal chitin. The Km values of BjCHI1, 2 and 3 for CM-chitin-RBV were estimated as 0.799mg/ml, 0.544mg/ml and 0.793mg/ml, respectively. When the colloidal chitin was used as substrate, the Km values were 0.2811mg/ml, 0.3883mg/ml and 1.6431mg/ml, respectively，indicating chitin-binding domain can increase affinity of chitinase to insoluble substrate. In the agglutination activity assay, only BjCHI1 shows activity when the protein concentration was more than 33ug/ml, while BjCHI2 and BjCHI3 without agglutination activity even when the concentration was increased as high as 800ug/ml. This means that the two chitin-binding domains in BjCHI1 are essential for agglutination and BjCHI1 is the first protein which shows both chitinase and agglutination activity identified so far in plants..The plasmid pCB302-117 was constructed by inserting the full-length of BjCHI1 gene into the SmaI site of the minibinary vector pCB302-1. Thus BjCHI1 and BjCHI2 genes were transferred into tomato varieties Zaofen No.2 mediaed by Agrobacterium tumefaciens. Disease resistance was tested with Fusarium oxysporum on R1 generation of transgenic plants. Two transgenic lines showed enhanced resistance to the fungus.

植物几丁质酶与抗真菌病害有关。芥菜几丁质酶基因BjCHI1编码的蛋白质具两个几丁质结合域，其衍生克隆BjCHI28编码的蛋白质只有一个几丁质结合域。将这两个基因转入番茄并表铮ü舛ū泶锏鞍字实募付≈拭富钚约袄胩逡志笛楹图ㄗ蛑仓甑目共⌒裕赏寮付≈式岷嫌蚴坑爰付≈拭腹δ艿墓叵怠６愿没虻睦镁哂兄匾庖濉