Genetic Analysis Of GhMYB Like2 In The Transgenic Cotton(Gossypium Hirsutum)progeny And Its Functional Characterization

In China,cotton is widely cultivated as an important economic crop.It is of great significance to study the molecular regulation mechanism of cotton fiber development and improve the yield and quality of cotton fiber through transgenic technology.Cotton fiber development is affected by the external environment and endogenous hormones.Previous studies have shown that brassinolide(BR)plays an important role in cotton fiber development.As a transcription factor widely distributed in plants,MYB transcription factors play a vital role in the regulation of cotton fiber growth and development.We used yeast two-hybrid technique to screen cotton fiber cDNA library and identified a protein which interacts with cotton 14-3-3e protein,named GhMYB Like2.In this paper,the genetic phenotypes and related molecular mechanisms of GhMYB Like2 transgenic cotton progeny lines were studied.The main results obtained are as follows:1.Subcellular localization analysis of GhMYB Like2 proteinThe ORF of the GhMYB Like2 gene is 1086 bp long which encoding 361 amino acids.There is a MYB-DNA-binding region not only between the 16th and 61st amino acid sequences,but aiso between the 70th and 112th amino acid sequences.That is to say it GhMYB Like2 belongs to the R2R3-MYB protein.In order to explore the subcellular localization of GhMYB Like2 protein in vivo,a fusion expression vector of GhMYB Like2 and eGFP was constructed and transferred into Agrobacterium GV3101 strain cells.The Agrobacterium liquid was injected into the epidermis of young tobacco leaves.After 2-3 days of culture,the young leaves of the tobacco leaves were removed.under the confocal microscope.GFP fluorescence was observed mainly in the nucleus,which proved that GhMYB The Like2 protein is localized to the nucleus and may be a transcriptional regulator.2.Genetic analysis in the GhMYB Like2 progeny transgenic cottonTo explore the function of the GhMYB Like2 gene in cotton fiber development,genetic analysis was performed in T2 and T3 GhMYB Like2 RNAi transgenic cotton.The cotton fiber RNA extracted from 9 days after flowering was detected by real-time quantitative RT-PCR.Compared with the wild type,the expression level of GhMYB Like2 in RNAi transgenic cotton fiber was significantly down-regulated.The ovules of GhMYB Like2 RNAi transgenic cotton on the day of flowering were observed using the hand-slicing technique.It was found that GhMYB Like2 RNAi transgenic cotton fiber cells protrusion slowly.GhMYB Like2 RNAi transgenic cotton plants showed no significant difference in leaf morphology,cotton size and mature seed size compared with wild type plants,indicating that GhMYB Like2 gene silencing did not affect the agronomic traits of cotton plants.The length of mature fiber and fiber quality of GhMYB Like2 RNAi transgenic cotton were measured.It was found that the mature fiber length of GhMYB Like2 RNAi transgenic cotton was significantly shorter.Compared with the wild type,the length of cotton fiber of several RNAi lines decreased 10.33%,9.58%,8.31%,5.57%,10.42%and 10.99%,respectively,while the breaking specific strength decreased,the micronaire value increased.This results show that GhMYB Like2 plays an important regulatory role in cotton fiber development.3.Transcriptome analysis of GhMYB Like2 RNAi transgenic cotton fiberGhMYB Like2 RNAi transgenic cotton and wild-type cotton fiber RNA were extracted from 9 days after flowering for transcriptome sequencing analysis.The results showed that there were 1451 genes with significant changes in the expression level of RNAi line 1 compared with wild type,of which 766 genes were up-regulated and 688 genes were down-regulated;the expression level of RNAi line 2 changed significantly.There are 767 genes,of which 601 are up-regulated and 166 are down-regulated.Among the genes whose expression levels significantly changed,there were 123 genes shared by RNAi line 1 and 2.Analysis of the differential genes in the cotton fiber of wild-type and two RNAi transgenic lines,revealed that there were significant changes in the expression of 24 BR-related genes.Among them,the expression of 5 genes was significantly up-regulated in GhMYB Like2 RNAi transgenic cotton,indicating that GhMYB Like2 may be an important regulator of BR signaling pathway.The differentially expressed genes were classified by the KEGG bioinformatics database,and the genes for these changes were mainly encoded by some transcription factors,oxidoreductases,cellulose synthase,lignin synthetase,etc.most of them may regulate cell elongation and differentiation.In addition,these differentially expressed genes were also found to be involved in plant hormone signaling,lignin biosynthesis and metabolism,flavonoid biosynthesis and metabolism,phenylalanine biosynthesis and metabolism,heat shock protein biosynthesis and metabolism,and transcriptional regulation redox and catalytic processes,indicating that GhMYB Like2 plays an important regulatory role in cotton fiber development.4.GhMYB Like2 and GhMPK6/9 protein interaction analysisIt has been reported that MYB transcription factors are often phosphorylated by MPK protein kinases thereby exerting their regulatory functions.It was found that GhMYB Like2 and some GhMAPKs were co-expressed during cotton fiber development,and the expression patterns of GhMPK3,GhMPK5,GhMPK6,GhMPK9 and GhMPK23 in cotton fibers were similar to those of GhMYB Like2.Therefore,it is speculated that the GhMYB Like2 protein may interact with these GhMPKs proteins to be phosphorylated to function.Protein interaction analysis using bimolecular fluorescence complementation confirmed that GhMYB Like2 interacts with GhMPK6 and GhMPK9.At the same time,it was further proved that GhMYB Like2 interacts with GhMPK6 and GhMPK9 by in vitro pull-down experiments.