Analysing Of GaMEKK And GaMPK4by VIGS Method In Gossypium Barbadense

Mitogen-actived protein kinase (MAPK) is the Ser/Thr protein kinase whichexists in eukaryotes. MAPK cascades are one of the most important signaltransductions in eukaryotes, and mainly mediate the signal transduction of response tostress in plants. MAPKKK (MEKK), MAPKK (MKK/MEK) and MAPK compose theMAPK cascades, and transmit the signal successively by phosphorylation fromupstream to downstream.In recent years, some studies showed that the signal transmission of plantresponse to pathogen are bound up with MAPK cascades, and had set up some MAPKcascades related to plant response to pathogen in variety of plants (Such asArabidopsis, Rice, Tobacco, etc). In this study, island cotton variety3-79was used asresearching material; the partial sequences of GaMEKK and GaMPK4were cloned.And the two gene functions were further dissected by VIGS method.Virus induced gene silencing is a technology. It is refer to the virul vector carrythe plant functional gene to infect the plant, in order to the gene was silenced and thephenotype was changed, and then studying the function of target gene.The results were shown as following:1. Using BLAST searching for island cotton MEKK and MPK4gene according tohomologs of Arabidopsis MEKK1and MPK4genes on NCBI, their partial sequenceswere cloned, respectively349bp sequence of GaMEKK and340bp sequence ofGaMPK4, the specific primers designed by DNAMAN. And total RNA from theisland cotton var.3-79was reverse transcripted to get the cDNA, which was used astemplate for obtaining fragments by PCR amplification. The fragments wererespectively inserted into the vector pYL-156, which were then transformed intoE.coli DH5α. The result of sequencing analysis confirmed that the amplified productis the target gene.2. The plasmids of pYL-156-GaMEKK and pYL-156-GaMPK4were respective-ly introduced into Agrobacterium tumefaciens GV3101to obtain the engineeringbacteria of VIGS. Inoculating engineering bacteria into the cotyledons of cotton seedling without true leaves. Eventually15RNAi plants of GaMEKK gene and15RNAi plants of GaMPK4gene was developed.3. Detecting the effect of the GaMEKK and GaMPK4gene knockdown by VIGS.When the control leaves of plants with PDS gene silence appeared white spot, theleaves from the plants with target gene silence and control were collected for RNAextraction. And reverse transcription and PCR were performed for confirming RNAiefficiency. The results showed that the RNA expression level of GaMEKK andGaMPK4were suppressed in VIGS plants.4. It was found that the VIGS plants with GaMEKK or GaMPK4RNAi inoculate-ed the Pseudomonas syringae pv.tomoto DC3000and Verticillium dahliae991exhibited lower resistence compared with control. And the marker genes corcerningSA and JA pathway related to response to pathogen were monitored, the resultsshowed that in VIGS plants, the expressive level of genes regarding to JA(LOX1,OPR3, JAZ6) is lower than those of control, while the genes associated SA(NPR1,PR5) showed higher than control.