| Cotton is an important economic crop and an important source of natural fibers and oilseeds in China and the world.Cotton yield and quality will be affected by various nonbiological adversities.At present,drought,salinity and low temperature limit cotton growth,which are the main factors of abiotic stresses.The yield of cotton and other crops were seriously reduced by abiotic stresses.Water absorption and distribution in cells were limited,that causes water imbalance and restrict growth and development.Therefore,it is of great significance to study the adaptability of cotton to abiotic stress and cultivate new varieties of stress-resistant cotton.Soybean GmTIP2;3 is a typical Aquaporins(AQPs),belongs to the ancient MIP family.MIP family is a type of integrated membrane proteins that efficiently and specifically transport water molecules.Since Arabidopsis and soybean plants overexpressing GmTIP2;3 showed much resistant to drought compared with wild type.Overexpression of GmTIP2;3 in cotton was expected to improve resistant to drought.The CRISPR/Cas9 system is the latest genome editing technology that has emerged in recent years.In CRISPRCas9 system,the Cas9 nuclease induces a double-stranded DNA break(DSB)at a specific target site,which was specified by the sgRNA sequence.DSB will repaired by two pathways,Non-homologous end joining,which results in stochastic insertions and deletions,and homozygous recombination,which may cause homozygous mutants.CRISPR/Cas9 system was introduced into cotton by Agrobacterium mediated transformation and injection of cotyledons.For the purpose of optimizing transformation method and creating new germplasm,two aspects of research were carried out in this study:1)Agrobacterium-mediated hypocotyl genetic transformation method was used to obtain GmTIP2;3 overexpressing transgenic cotton that may improve resistance for drought;2)Using particle bombardment transform CRISPR/Cas9 system into embryogenic calli,cotton,in which gland formation genes were mutated,were generated as new germplasm for low phenol cotton.The main findings are as follows:1.Creation of a new transgenic cotton overexpressing soybean GmTIP2;3.BLAST analysis of GmTIP2;3 showed the upland cotton GhTIP2;1 has the highest identity with GmTIP2;3.Alignment result showed that the amino acid sequence homology of GhTIP2;1At and the gene is 84.27%,and the amino acid homology of GhTIP2;1-Dt and the gene is 85.08%.To determine whether GhTIP2;1 related to drought,drought-induced transcriptome analysis was performed.The result indicated that the function of GhTIP2;1 was less related to drought resistance.Previous studies have shown that transgenic Arabidopsis and soybean overexpressing GmTIP2;3 both show significant drought resistance.Therefore,we constructed soybean GmTIP2;3 over-expression vector and transformed cotton by using Agrobacterium-mediated transformation to create cotton materials with improved drought resistance.To determine positive events in transgenic T0 generation plants,PCR amplification was performed with Promoter-gene specific primers.Nine positive clones were selected for further cultivation of pure lines.2.GhGl2 and GhGl3,genes related to gland formation,were edited by CRISPR/Cas9 system.For simplifying transformation purpose,we sought to test the possibility of particle bombardment transformation of CRISPR/Cas9 into embryogenic calli.According to previous report,GhGl2 and GhGl3 was selected as genes of interest.Target sites within exons of genomic sequences were selected and evaluated on the website of http://crispr.hzau.edu.cn/CRISPR/.Three sgRNA targeting sites were selected at 120bp,575bp and 345bp,were constructed and named as pKSE401::sgRNA1,pKSE401::sgRNA2 and pKSE401::sgRNA3.Using cotton embryogenic callus as the recipient,three constructs were transformed into cotton respectively by gene gun transformation.Given the observation of phenotype,genome edited plants were classified into 4 classes.The relative expression level of genes was determined by qRT-PCR.The result of gossypol content measurement by HPLC is consistent with phenotype and gene expression.Among them,the transgenic T0 plant 2-5 of pKSE401::sgRNA2 showed no glands and it was found that both GhGl2 and GhGl3 had two base deletions in the plant.Furthermore,high-throughput sequencing technology was used to detect potential off-target loci,and no off-target effect was found.The PCR assay of At-U6 promoter and Cas9 showed that presence rate of Cas9 is 100%.According to above results,gene gun based CRISPR/Cas9 system could realize gene editing in cotton,which provides a rapid and simple alternative method for creating genome edited cotton plants by CRISPR/Cas9 system. |
