Evaluation Of Fusarium Wilt Resistance Of GbCHI,GbDFR And GbF3’H Genes Of Gossypium Barbadense

Cotton is one of the most important cash crops in the world’s oilseeds and raw materials for the textile industry,and plays an important role in the national economy.Xinjiang is the main production area of Gossypium barbadense.In recent years,due to the increasing severity of Fusarium wilt,it has seriously affected the yield and fiber quality of G.barbadense.Early cotton fusarium wilt research focused on Gossypium hirsutum,but less research on G.barbadense.In this study,the key genes of flavonoid metabolism pathways in G.barbadense,GbCHI(GB_A13G0219.1),GbDFR(GB_A05G1949.1),and GbF3’H(GB_D07G1307.1),were related to disease resistance through preliminary transcriptome data and disease resistance expression profile data.The G.barbadense resistance material 06-146 was used as the research object.The VIGS technology was used to silence the G.barbadense GbF3’H gene,and the G.barbadense plants that had silenced the GbF3’H gene were obtained.The GbCHI,GbDFR and GbF3’H genes were silenced in the G.barbadense plant.At the same time,the overexpression vector pCAMBIA3301-GbCHI of GbCHI gene was also constructed,and transgenic plants were obtained by Agrobacterium pollen tube channel method.In this paper,VIGS technology was used for transient silencing and Agrobacterium pollen tube channel method for genetic transformation of G.barbadense,and the resistance of GbCHI,GbDFR and GbF3’H genes,key genes of the flavonoid metabolism pathway in G.barbadense,to Fusarium wilt was studied.The main results are as follows:(1)Based on the previous transcriptome sequencing,this experiment analyzed the key genes GbCHI,GbDFR and GbF3’H of the flavonoid metabolism pathway of G.barbadense and related to disease resistance.GbF3’H gene was amplified and used to construct the required fragment of VIGS silencing vector.Virus-induced gene silencing(VIGS)technology was used to obtain GbF3’H silencing plants in G.barbadense.The cotton plants were injected with virus.After 15 days,the roots,stems,and leaves of the empty control group and the silent group were taken,and three plants in each group were used to form a mixed pool.Analysis of gene silencing in each treated sample by real-time fluorescent quantitative PCR,inoculation of Fusarium wilt.The spot area is large.The expression levels of GbF3’H gene in roots,stems and leaves were lower in G.barbadense plants than in the empty control group.The effect of GbF3’H gene silencing was:leaf>root>stem.The disease index sizes were:wild type(WT):15.75<empty vector control group(TRV::00):15.9<TRV2-GbF3’H:29.5.The incidence of cotton seedlings injected with silenced genes was higher than that of empty vector control group serious.(2)Mix the suspensions of TRV2-GbCHI,TRV2-GbDFR and TRV2-GbF3’H vectors to silence the three genes GbCHI,GbDFR and GbF3’H.Take the empty control after 15 days of virus injection.The roots,stems,and leaves of cotton plants in the group and the co-silence group were composed of 3 plants in each group.The status of gene silencing in each treated sample was analyzed by real-time fluorescence quantitative PCR,the expression level of each gene,and the silence were analyzed.Changes in the resistance of the treated materials to Fusarium wilt.The results showed that in cotton that had been silenced with TRV::F3’H,TRV::DFR,and TRV::CHI,the expression levels of GbCHI,GbDFR and GbF3’H genes in roots,stems,and leaves were lower than those in the empty control group,respectively.Among them,the silencing effect of GbCHI gene in G.barbadense is leaf>stem>root;the silencing effect of GbDFR gene in G.barbadense is leaf>root>stem;the silencing effect of GbF3’H gene in G.barbadense is leaf>root>stem.The cotton plants were inoculated with Fusarium wilt for 15 days after the cotton plants were injected with the virus.Investigation of the disease after 25 days showed that the incidence of cotton seedlings injected with the silenced gene was more severe than that of the empty control cotton seedlings.The disease index investigation the results showed wild type(WT):15.75<empty vector control group(TRV::00):15.9<TRV2-GbF3’H:29.5<TRV2-GbF3’H,TRV2-GbCHI,and TRV2-GbDFR co-infection:39.9.which indicates the resistance of co-silencing materials to Fusarium wilt was significantly lower than that of single gene silencing materials,and the resistance of single gene silencing materials was significantly lower than those of the empty vector control and wild type.The results showed that GbF3’H gene had a certain effect on the resistance of G.barbadense to Fusarium wilt,moreover,synergistic effect with GbCHI and GbDFR genes to enhance disease resistance.These results provide a reference for studying functional genomics of G.barbadense.(3)Using the G.barbadense disease-resistant variety 06-146 as the recipient material,an over-expression pCAMBIA3301-GbCHI vector of GbCHI gene was constructed,which was genetically transformed by the pollen tube pathway method mediated by Agrobacterium,and sprayed in the field.Screening and molecular testing of herbicide resistance,T2 generation G.barbadense plants transgenic with GbCHI gene were obtained.The transgenic G.barbadense and wild type G.barbadense plants were inoculated with Fusarium wilt.The statistical results showed that wild type G.barbadense inoculated with Fusarium wilt is more severe than inoculated with Fusarium wilt in transgenic G.barbadense.The disease index investigation the results showed,transgenic G.barbadense:29.58;wild type G.barbadense:40.42.The results preliminary confirmed that the GbCHI gene played a role in the resistance of G.barbadense to Fusarium wilt.