Study On The Regulatory Function Of GhHOX4 In Fiber Cell Initiation And Elongation Of Cotton

Cotton is one of the most important fiber crops in the world,and biologists and breeders have been using modern biotechnology and methods to improve cotton fiber quality and increase cotton yield.Cotton fiber is a single-celled epidermal hair derived from the polar elongation of cotton ovule epidermal cells,which is similar to the developmental pattern of Arabidopsis epidermal hair.Previous studies have shown that the Arabidopsis GLABRA2(AtGL2)gene plays an important role in the differentiation of Arabidopsis leaf epidermis and root epidermal hair cells.By homologous sequence alignment,we found that cotton GhHOXl,GhHOX2,GhHOX3,GhHOX4 and AtGL2 have the same Homeobox domain,and the amino acid homology reached 95%,71%,72%and 63%,respectively.In the previous work,the GhHOX4 gene was cloned and identified from Upland cotton,and its function and molecular mechanism in Arabidopsis were studied.Based on the results of previous studies,this study continued to study the length of GhHOX4 transgenic Arabidopsis epidermis;and obtained the genetic phenotype analysis and fiber of T2,T3 and T4 generations of GhHOX4 RNAi transgenic cotton and GhHOX4 OE transgenic cotton.Quality detection,transcriptome analysis of cotton fibers in GhHOX4 transgenic cotton 6 days after flowering by RNA-Seq technique,and the regulation of GhHOX4 in the initiation and elongation of cotton fibers was analyzed.The main results obtained are as follows:1.GhHOX4 regulates the initiation and elongation of cotton fiber cellsIn order to study the function of GhHOX4 gene in cotton fiber development,we obtained GhHOX4 overexpression and RNAi transgenic cotton plants by Agrobacterium-mediated cotton transformation.Different generations of GhHOX4 transgenic cotton progeny lines(T1-T4 generation)were continuously planted,and the genetic phenotype of the transgenic cotton progeny lines was observed and analyzed.The results showed that the expression of GhHOX4 was significantly increased in GhHOX4 overexpressing transgenic cotton fibers,while the expression of GhHOX4 was significantly decreased in GhHOX4 RNAi cotton fibers.The expression levels of this gene in T2,T3 and T4 generation GhHOX4 overexpression and RNAi transgenic cotton fibers were also consistent with the T1 generation analysis.The length of mature cotton fiber of transgenic cotton was statistically analyzed.The results showed that the mature fiber of GhHOX4 overexpressing transgenic cotton was significantly longer than that of wild type,while the GhHOX4 RNAi transgenic cotton fiber was significantly shorter.In addition,GhHOX4 transgenic cotton ovule in vitro culture experiments were carried out,and the results were also consistent with the statistical analysis results of mature fibers.The above results indicate that the GhHOX4 gene plays a positive regulatory role in the elongation phase of cotton fibers.Using the hand-slicing technique,the ovule surface fibroblast processes of GhHOX4 OE and RNAi transgenic cotton lines and wild-type plants at 0,1,and 2 days after flowering were observed,and it was found that the progenitor cells on the surface of the ovule of GhHOX4 OE strain were more wild.The type cells were longer and more sparse,while the GhHOX4 RNAi strain did not differ significantly from the wild type.This suggests that the GhHOX4 gene may affect early initiation of differentiation and subsequent elongational development of cotton fiber cells.The average length,uniformity,breaking strength and micronaire value of cotton fiber are several important indicators for measuring the quality of cotton fiber.From the results of cotton fiber quality test,it can be seen that the fiber length of GhHOX4 RNAi transgenic cotton is shortened by 4.1%,the fiber length of GhHOX4 OE transgenic cotton is 3.9%longer,and the uniformity of cotton fiber is higher than 84%.The specific strength of the break was that the GhHOX4 RNAi transgenic cotton became 7.6%weaker and the GhHOX4 OE transgenic cotton became 1.8%stronger.This indicates that the gene affects the formation of key quality traits such as cotton fiber length.2.GhHOX4 affects downstream gene expression during elongation and development of cotton fiberTo study the regulation of GhHOX4 gene expression in downstream genes in transgenic cotton fibers,we selected GhHOX4 RNAi cotton fiber RNA samples and wild-type RNA samples from the T2 generation 6 days after flowering,and performed RNA-seq sequencing analysis to screen possible cotton in upland cotton.Differentially expressed genes regulated by GhHOX4.Transcriptome analysis revealed that the expression of 453 genes in GhHOX4 RNAi transgenic cotton fibers was altered compared with wild type,of which 86 genes were up-regulated and 367 genes were down-regulated.These differentially expressed genes mainly contain some genes related to cell and organelle,as well as genes related to binding,catalytic activity,transport activity,molecular sensor activity,etc.,which may indicate that cells are actively metabolized during the initial differentiation and rapid elongation of cotton fiber cells.The material required for fiber elongation is rapidly synthesized and transported,thereby ensuring the continuous elongation of the fiber cells.Subsequently,combined with the heat map analysis of differential genes and the existing literature,some genes involved in cotton fiber development were found to be regulated by GhHOX4.Therefore,the results of transcriptome analysis indicated that the GhHOX4 gene may regulate the elongation and development of cotton fibers by regulating or affecting downstream gene expression during the initiation and elongation stages of cotton fibers.