Study On The Formation Mechanism And Gene Function Of Dwarfing Mutants In Upland Rice

Due to the high agronomic value of dwarf mutants,studying the molecular mechanism of plant dwarfing and exploring new sources of dwarfing is of great significance to the improvement of crop resource innovation and genetic breeding.This subject takes the upland rice transgenic line b33 as the research object.b33 is derived from Kunhan 1.It is a transgenic offspring obtained by transforming the gene LEA8981 from the late embryonic development rich protein family cloned from Physcomitrella patens into H1.We analyzed the phenotype,target gene insertion site and gene expression near the insertion site of b33,and the results showed that the expression of the downstream gene lnc RNA gene near the insertion site of the mutant exogenous gene may be the main cause of plant dwarfing.This study aims to use the gene knockout technology of the CRISPR/Cas system to obtain the T0 generation plants with the lnc RNA gene knocked out.Through the preliminary analysis of the phenotype of the T0 generation plants,it is explored whether lnc RNA actually affects the plant height of upland rice.The research content and experimental results were as follows:1.Using the online design website design(http://crispor.tefor.net/)to target sg RNAs at different target sites of lnc RNA.The sg RNA with specificity,comprehensive score,and low off-target rate was selected as the gene knockout target site,and a total of 6 target sites were screened.2.We connected the synthesized target site adaptor to the intermediate vector p Os-sg RNA by means of restriction enzyme digestion and ligation,and transfer it into E.coli DH5α.After PCR amplification of sg RNA,sequencing and comparison,it was verified whether the target adaptor site and the intermediate vector were connected correctly,and a total of 6 correctly constructed entry vectors were obtained.3.Using the LR reaction of Gateway technology,connect the entry vector to the target vector p H-Ubi-Cas9 to transform E.coli(DH5α).After resistance screening,PCR amplification of sg RNA and sequencing analysis,6 gene editing expression vectors were finally obtained.4.The gene editing expression vectors were transformed into Agrobacterium tumefaciens AGL-1 and transformed into H1 and b33 by Agrobacterium tumefaciens respectively,and several positive plants were obtained.5.The total of 23 T0 generation transgenic plants were obtained by genetic transformation for H1 gene editing,of which 3 plants were successfully edited for target site 1,and 16 plants for target site 2 were successfully edited.Total of 21 T0 transgenic plants were obtained by gene editing and transformation of b33,of which 5 plants were successfully edited at target site 1 and 12 plants were successfully edited at target site 2.6.The sequencing analysis results showed that the gene editing results of the target lnc RNA are mostly 1-10 bp base deletion mutations and large fragment deletion mutations greater than 300bp;base insertion mutations are mostly 1bp,2bp base insertions.The results showed that there was no significant difference in plant height between the target gene lnc RNA gene of H1 and the untransformed H1;in the b33 gene editing plants,the plant height of AB2-22 and AB2-29 plants were basically restored to the level of H1,with the deletion of 13 bp and 391 bp base fragments,respectively.It is speculated that the high expression of lnc RNA gene may be directly related to the dwarf phenotype of mutant b33.