Phenotypic Identification And Histochemical Analysis Of Transgenic Plants With Interference And Knockout GhCENH3 Gene In Gossypium Hirsutum L.

As an indispensable part of eukaryotic chromosome composition,centromere plays an irreplaceable role in mitosis and meiosis.CENH3(Central specific histone H3)gene,as a marker of functional centromere,can guide the correct pairing of homologous chromosomes,the correct binding of sister chromatids,and the correct separation of chromosomes during mitosis and meiosis,so as to ensure the correct division of fine cells and the normal development of plant reproduction.In this study,Gh CENH3 gene of upland cotton was cloned by homologous gene method,and the effect of Gh CENH3 gene on cotton cell division and plant growth and development was studied by interference and knockout,and the mechanism of knockout Gh CENH3 gene affecting cotton fertility was analyzed.The main findings are as follows:(1)Three Arabidopsis At CENH3 homologous genes were identified in upland cotton,which were named Gh CENH3-A,Gh CENH3-D and Gh H3.3 respectively.The nucleotide sequence consistency of Gh CENH3-D and Gh CENH3-A is 97.84%,which is a pair of homologous genes in tetraploid upland cotton.Subcellular localization shows that Gh CENH3 gene is mainly located in the nucleus.Gh CENH3 gene of different species has the characteristics of highly variable N terminal and highly conservative C terminal.(2)The Gh CENH3 gene interference vector was constructed and transferred into cotton,and a total of 7 positive transformation events were obtained.Compared with the wild type,the interference line C2 still has the phenotype of shorter plant height,smaller flowers,less pollen,lower boll setting,lower fiber quality in the T3 generation.At the same time,the expression of Gh CENH3 in the interference line C2 was significantly lower than that in the wild type at the three main stages of anther development.(3)The Gh CENH3 gene and Gh H3.3 gene knockout vectors were constructed and transferred into cotton,and two edited transformation events were obtained respectively.There was no significant change from the plant edited by Gh H3.3 to the representative type of T3,and the plants of the T3 generation lines edited by Gh CENH3,Ghcenh3-1-2-5and Ghcenh3-1-6-1,were shorter and thinner than the wild type plants,with lower boll setting,lower fiber quality and smaller seed development.Phenotypic identification and tissue section of plant line Ghcenh3-1-6-1 at different stages of anther development showed that cotton anther development was abnormal and pollen vitality was affected.(4)The Gh CENH3 gene interference strain appeared individual chromosome lag phenomenon in the late mitosis stage,and did not see the formation of micronucleus in the late mitosis stage.The Gh CENH3 gene knockout line showed unequal division at the end of the first meiotic division,two cells with unequal number of chromosomes were seen at the beginning of the second meiotic division,and some chromosomes lagged at the end of the second meiotic division.Finally,four sperm cells with unequal number and different size of chromosomes were formed at the end of the second meiotic division.(5)CUT&Tag analysis showed that the Ghcenh3 knockout mutant had more clean reads on different chromosomes,more open chromatin state,looser binding state of histone and DNA,more gene regulatory proteins and RNA polymerase combined with its DNA sequence,abnormal RNA transcription,and increased H3K4me3 level.(6)The hybrid of the strain Ghcenh3-1-6-1 with the recessive glandless material "YZ1" yielded 20 glandular athered materials,and the haploid induction rate was 1.85%.