Anti-verticillium Dahliae Protein Of Aspergillus Niger And Trichoderma Aculeatus In Cotton Rhizosphere

Gossypium Spp.is an economic crop that is widely planted worldwide,and the spread of Verticillium wilt has brought significant losses to cotton production.Found the protein components and genes with antibacterial effects,cultivating genetically modified disease-resistant cotton is one of the methods to control Verticillium wilt.The main purpose of this study is to isolate the anti-Verticillium dahliae proteins and genes from the rhizosphere fungi resistant to Verticillium dahliae.The main research contents and results are as follows:Using the dilution plate method,24 pairs of plants were isolated from the rhizosphere soil of 6 upland cotton varieties including Zhongmiansuo 08,Zhongmiansuo 41,Zhongzhimian 2,Ji Mian 11,Lumianyan 28 and Lumianyan 21.Verticillium dahliae Kleb.V1070 and Gibberella sp.F0609 are fungi with inhibitory effects.According to morphology and 18 S r DNA identification,it is found that the rhizosphere fungi of different varieties have no obvious differences.Mainly Aspergillus niger and Trichoderma sp.Among them,the average inhibitory rate of Aspergillus niger against V1070 is 91%,that of Trichoderma aculeatus is 87%,that of Mortierella alpina is 76%,and that of Fusarium laminum sp.is 70%.The fungi with good inhibitory effect on the V1070 strain were screened by the confrontation culture method,and they were identified as Aspergillus niger(An1)and Trichoderma aculeatus(Tr1).The two fungi have no effect on the development of Jimian 11,and An1 has a promoting effect on the growth of seedlings.Jimian 11 was cultivated with An1 mixed substrate,and V1070 was inoculated by root irrigation method.The control effect of An1 treatment group after inoculation was 23.5%.The fermentation broth of Aspergillus niger An1 and Trichoderma aculeatus Tr1 and the crude protein obtained by ammonium sulfate precipitation and salting-out method significantly inhibited V1070 spore germination and mycelial growth.The inhibitory rate of An1 crude protein aqueous solution on spores can be 100%,and the inhibitory rate of methanol solution is 65%.The crude protein in An1 and its fermentation broth can significantly increase the content of lactate dehydrogenase and active oxygen of V1070,the increase in chitin and ergosterol was not obvious.The inhibition of V1070 by An1 crude protein may be related to the damage of its cell membrane.Online search of the protein database of the fungus phylum compared the types and functions of proteins in Aspergillus niger An1 and Trichoderma Tr1,and identified 132 and86 proteins respectively,and found 24 proteins with potential antibacterial functions.According to bioinformatics analysis,1,3-β-glucosidase EGLC has a GH17 domain,α-1,2-mannosidase ASP1 has a GH47 domain,and β-xylanase ASP2 has a GH superfamily structure.Aspergillus peptide PEPA has a GH7-CBH super family domain,and cellobiohydrolase CBHA has a Pepsin＿retropepsin＿like super family domain,logical processes such as inhibiting the synthesis of pathogenic fungi’s cell wall effect.The Eglc gene cloned from Aspergillus niger An1 was prokaryotically expressed and purified.Its base number is 1329 bp and encodes 442 amino acids.The EGLC protein is closely related to the glycoside hydrolase of Aspergillus niger CBS 513.88.The Eglc gene was connected to the linear vector p BI121,which was transformed into Agrobacterium tumefaciens EHA105.Arabidopsis thaliana was infected by the dipping method to obtain T0 generation plants,and T1 and T2 generation transgenic seedlings were obtained by screening with resistant plates.After the seedlings were inoculated with Verticillium dahliae V1070 strain by root irrigation method,it was found that the disease index of wild-type Arabidopsis thaliana was 78.5,the disease index of the transgenic plants was 61,and the control effect reached 22.3%.It shows that the product of Eglc gene expression has an inhibitory effect on the infection of V1070.This study found that Aspergillus niger An1 has significant control effect on cotton verticillium wilt.The fermentation broth of Aspergillus niger An1 and Trichoderma aculeatus Tr1 contains anti-Verticillium wilt protein,and the Eglc gene can be used as an anti-Verticillium wilt gene.