Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.

Cloning and Expression of a Chitinase Gene from Serratia marcescens Strain C8-8

A 1 692 bp long chitinase-encoding ch/A gene was cloned from the genomic DNA of Serrat/a marcescens strain C8-8 by PCR, which was speculated to en- code a 563 aa long polypeptide chain with molecular weight of about 60.9 kD. Homolog analysis showed that the chiA gene sequence cloned from C8-8 shared the highest similarity with cMA sequences from Serrat/a maresscens strains 141 （ DQ 990373.1 ） and 14041 （ DQ 493896. 1 ）, which reached 99%. Domain analysis showed that N-termlnal （23 aa） of the chiA gene cloned from C8-8 harbored typical signal peptide sequence, while C-telminal harbored the other two domains, in- eluding the PKD region （73 aa） and chitinase catalytic region （387 aa）. The PCR fragment was digested with restriction endonucleases and cloned into plasmid pET28a. The recombinant plasmid pET＇28a-ch/A was firstly transformed into Escherichia coli DI-I5 , and then transformed into expression host E. coli DH3 to express ch/A gene. The recombinant strain DH3 chiA could produce transparent hydrolysis circles on the colloidal chitin plate induced by isopropyl-l-thiogalactopyranoside （IFrG）. SDS-PAGE electrophoresis analysis showed that, a protein with relative molecular weight of about 60 kD was expressed by the recombinant strain DH3 chiA, which was consistent with the except molecular weight. After initial purification, biological activity test showed that the recombinant expression product could hydrolyze chitin, which produced transparent hydrolysis circles on the colloidal chitin plates. Results indicated that chiA gene from Serrat/a marcescens strain C8-8 had biological functions and could be utilized as a potential biological control factor.

Molecular cloning and primary sequence analysis of a gene encoding a putative chitinase gene in Brassica oleracea var.capitata

Chitinase, which catalyzes the hydrolysis of the β-1,4-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin, is involved in inducible plants defense system. By construction of cabbage (Brassica oleracea var.capitata) genomic library and screening the library with pRCH8, a probe of rice chitinase gene fragment, a chitinase genomic sequence was isolated. The complete nucleotide sequence of the putative cabbage chitinase gene (cabch29) was determined, with its longest open reading frame (ORF)encoding a polypeptide of 413 aa. This polypeptide consists of a 21 aa N-terminal signal peptide,two chitin-binding domains different from those of other classes of plant chitinases, and a catalytic domain. Homology analysis illustrated that this cabch29 gene has 58.8% identity at the nucleotide level with the pRCH8 ORF probe and has 50% identity at the amino acid level with the catalytic domains of chitinase from bean,maize and sugar beet. Meanwhile,several kinds of cis- elements,such as TAT.A box, CAAT box, GATA motif, ASF-1 binding site, wound-response elements and AATAAA, have also been discovered in the flanking region of cabch29 gene.

Genetic transformation ofpopulusxeuramericana cv. guariento with chitinase gene

In this paper, populus xeuramericana cv. Guariento was transformed with bean chitinase by Agrobacterium tumefaciens-mediated leaf disc method. Firstly, the leaf explants were pre-cultured at 25℃ for 2d. Secondly, they were infected in Agrobacterium tumefaciens suspension （OD600=0.5） for 20 rain, then were co-cultured for 3d in the dark. Thirdly, the explants were transferred to the selection culture medium （containing Kanamycin 40 mg.L^-1 and Cefotaxime Sodium 800 mg-L1） and incubated at 25℃ until resistance buds formed. Chitinase activity was determined for the positive plants by PCR and PCR-Southern blot hybridization analysis. And, chitinase activity of positive plants was significantly higher than that of control plant, and the highest ratio of activity of NO.4 to that of control was 3.41. It showed that bean chitinase gene had been expressed in the plant genome.

A Novel Cotton Gene Encoding a New Class of Chitinase

DA novel chitinase gene (GhChia7) was isolated from salicylic acid (SA)-treated cotton cotyledons and characterized by DNA sequence analysis of its cDNA and genomic DNA clone. The deduced amino acid sequence, designated as class VII chitinase, shares about 30% identity to class I or II chitinases, and does not correspond to any of the previously characterized classes I-VI chitinases. Northern blotting analysis showed that the transcripts of GhChia7 were abundant both in cotton fibers and in the roots of the seedlings. The accumulation of GhChia7 mRNA in SA-treated cotyledons reached maximum at 7.5 mmol/ L concentration after 18 h. Results indicate that GhChia7 might play an important role in cotton's active defense response.

Characteristics of Resistance to Rice Sheath Blight of Zhongda 2,a Transgenic Rice Line as Modified by Gene “RC24”

The transgenic rice, Zhongda 2, which was genetically modified from an indica rice line Zhuxian B by rice chitinase gene (RC24), had high resistance to rice sheath blight (Rhizoctonia solan!) in laboratory and a two-year field experiment. The pathogen could invade sheath of Zhongda 2 and induce symptoms of the disease. No difference was noted in time of penetration or incubation period between Zhongda 2 and non-transgenic rice control, Zhuxian B, but the hyphae lysate could be observed earlier than control. Its resistance expressed as to inhibit the growth of mycelium in host tissue. Fis from Zhongda 2(4) crossed with other five non-transgenic rice lines showed higher resistance than donor non-transgenic parents, but the resistance was different along with the different maternal parents.

Functional properties of a chitinase promoter from cabbage (Brassica oleracea var. capitata)

The 5’-region of the chitinase gene cabch29, derived from Brassica oleracea var. capitata, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5’-deletion fragments were linked to reporter geneβ-glucuronidase (GUS) as translational fusions, and the expression of these chimeric genes was analyzed in vegetative organs and tissues. Sequences up to -651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues. The addition of further upstream sequences (-651 to -1284) enhanced expression level, and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding. Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-GUS fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment. The location of GUS activity appears to be cell-specific, being highest in vascular cells and epidermal cells of stem, leaf and roots. Meanwhile, the temporal and spatial expression of cabch29-GUS fusion gene has been investigated. Among the different vegetative organs, a high level of GUS activity was observed in stem and a moderate one in roots;whereas, wounding stress led to a high level of GUS in stem and moderate one in leaf.

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world. However, it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. In this study, the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique. The lines were the T2 of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene, and were positive by Southern Blot analysis. The resistance difference was studied through comparing the death percentage of transgenic soybean with the control. The results showed that four lines were more resistant to P sojae, whereas other one had no significant difference in comparison with the control. These transgenic soybean lines with enhanced resistance to P sojae will be useful in soybean resistance breeding.