Analysis And Functional Identification Of Genes Related To Flowering Time Of Cotton

Cotton is one of the most important economic crops in the world.The production of cottonseed oil and the supply of fibers have extraordinary significance for industry and commerce.In China,cotton is also an important agricultural economic crop.However,based on the national conditions of China's arable land area and large population base,the cotton planting area is extremely limited,and the conflict between grain and cotton is prominent.Flowering means that the plant begins to take reproductive growth as the growth center,and early flowering can promote early maturity and shorten the growth period of the plant.Therefore,starting from the perspective of cotton flowering regulation,overcoming the key mechanism of cotton flowering regulation,and realizing the cultivation of high-quality cotton in short-season cotton is one of the important methods to ease the current land situation,optimize agricultural planting structure,and achieve high yield and high efficiency.Previous studies have shown that flowering regulation has been gradually studied to analyze multiple regulatory pathways,each of which has its key genes.For example,the FLC gene is one of the key genes in the vernalization pathway,which plays a negative role in regulating flower formation;It has important significance in the photoperiod pathway,which is the maintenance of the external photoperiod changes and the plant's own flowering regulation.This experiment started from the critical period of cotton flower primordia,and carried out face-to-point through transcriptome sequencing,overexpression technology,CRISPR / Cas9 gene editing technology,matting yeast library screening test technology and subcellular location technology.Analysis and research,the main research results are as follows:(1)By dividing the period of flower bud formation into four stages: undifferentiated flower bud,imminent differentiation,initiation of differentiation,and differentiation completion,using transcriptome sequencing technology to focus on the dynamic progress and evolution of transcriptome during flower bud formation.It is found that flower bud differentiation affects a large number of differential changes in genes from the beginning to the end,revealing that flower bud regulation is a complicated process;GO annotation analysis showed that genes related to gene expression,redox and transcription factors were related to flower buds The differentiation is closely related;KEGG analysis found that genes in the pathways of plant hormone signal transduction,circadian rhythm,cytochrome P450,etc.are likely to be closely related to the regulation of the floral primordia differentiation process;The variable shear event has a specific performance in the process of flower bud differentiation,which is likely to participate in the flower bud differentiation and play an important role;(2)Further analysis of differential gene expression and annotation analysis information revealed that a family of auxin-responsive genes is likely to play a positive role in flower bud initiation.A member of his family was selected as an important candidate gene,and an unknown gene that may also have a positive function was also selected as a candidate gene.Quantitative PCR results showed that auxin-responsive genes are expressed in apical meristems,leaves,and stems,and the amount of expression decreases sequentially.Unknown genes are only detected in apical meristems.The expression of auxin-responsive genes is not only up-regulated with the up-regulation of auxin,but also may cross over with light regulation pathways.The expression of unknown genes is not affected by auxin and light.(3)In order to study and analyze the functions of the two candidate genes,from the perspective of overexpression experiment technology,by successfully cloning the full length of the candidate gene,complete the construction of the overexpression vector,and complete the field cotton plant and growth indoor Arabidopsis Conversion work.Each candidate gene harvested about 5000 T1 cotton seeds(5135 of auxin-responsive genes,4788 of unknown genes)and about 30000-40000 Arabidopsis seeds.These seeds are used in the subsequent screening test of positive seedlings,in order to verify that the candidate genes have the function of positively regulating flowering by observing whether the positive seedlings have a phenotype of early flowering.(4)By editing the candidate genes and changing the original gene function,the original gene function is reversely guessed.Using CRISPR / Cas9 gene editing technology,first target selection and primer design.Finally,a double-target gene editing vector was constructed,and field transformation of cotton plants was completed.Each candidate gene harvested about 7000-8000 cotton seeds(7210 of auxin-responsive genes,8341 of unknown genes)of T1 generation.These seeds will be used in subsequent screening experiments,the purpose of which is to verify that the candidate genes have the function of positively regulating flowering by observing whether the positive seedlings have a delayed flowering phenotype.(5)The matting method yeast two-hybrid test was used to screen the library to obtain the interaction protein information.The experimental results showed that in the end,neither of the two genes had been screened for the interaction protein.Based on the results of self-activation and toxicity detection,the quality of the AD library,the binding efficiency of the yeast cells in the experiment,etc.,the existence of experimental reagents and operating techniques and other problems are excluded,and the results are reliable.(6)The protein distribution of candidate genes was verified by experiments.Through subcellular localization experiments,it was found that both candidate genes may be membrane localization proteins.Therefore,the protein distribution and function of the two candidate genes may be the cell membrane.