Construction Of Multiple Mutants Of Arabidopsis IQM Gene Family And Its Phenotypic Analysis

The IQM family consists of 6 members,from IQM1 to IQM6,which are Ca²⁺-independent calmodulin-binding proteins?Ca MBP?.In recent years,our research group has conducted in-depth research on the functions of IQM family genes.Due to the lack of corresponding mutants,however,the growth and development changes caused by simultaneous mutation of multiple genes in this family have not been studied.In this study,therefore,the CRISPR/Cas9 genome editing technology was used to construct multi-gene mutants of the IQM family,and preliminary phenotypic analysis was conducted to obtain the following main results:1.Target adapters were designed on the genomes of 6 members of the IQM family,respectively,and constructed into corresponding small vector sg RNA expression boxes through homologous recombination:Laz At U3d-At U3b-At U3d-At U3b-At U6-1-At U6-29,which were integrated into p YLCRISPR/Cas9pubi-N expression vectors and then transferred into wild type Arabidopsis Col-0.Through antibiotic-resistance screening,70T₀generation plants were obtained.At last,a tetra mutant strain iqm?1,2,3,4?-1 of IQM1?IQM4 was obtained by sequencing the edited target.2.Through the following preliminary phenotypic analysis and related gene expression analysis of iqm?1,2,3,4?-1,the main results are displayed as following:1)The effect of sugar deficiency stress on seed germination:There was no significant difference between iqm?1,2,3,4?-1 and Col when germinating in 1/2 MS medium.However,the germination ratio of iqm?1,2,3,4?-1 was significantly higher than Col when germinating in sugar-free 1/2MS medium from day 2 to day 7.It is speculated that the seed germination of iqm?1,2,3,4?-1 is less sensitive to sugar deficiency than Col.Moreover,iqm?1,2,3,4?-1 showed no significant difference to Col when germinating in 1/2 MS medium without V_B5.2)The effect of sugar deficiency stress on root length:The root length of iqm?1,2,3,4?-1 was shorter than that of Col grown in 1/2 MS medium,and there were significant differences on day 4 and 5.Grown in the sugar-free vertical medium,the root length of iqm?1,2,3,4?-1 was close to that of Col,It indicates that the root growth of iqm?1,2,3,4?-1 is not sensitive to sugar deficiency.3)Effect of paclobutrazol?PAC?on seed germination:PAC is an inhibitor of plant hormone gibberella?GA?synthesis.When PAC was added in 1/2 MS medium,the germination ratio of the tetra mutants was significantly lower than that of wild type.When PAC and GA3 were added to 1/2 MS medium,the germination ratio of Col almost recovered to that of CK?blank control group?,but the germination ratio of iqm?1,2,3,4?-1 could not completely recover,indicating that it was more sensitive to the increase and decrease of GA than Col.The phenotypes of 4 related single mutants in the above two treatments were observed respectively.Only iqm3-2 showed a phenotype consistent with that of the tetra mutants,as indicated above,iqm?1,2,3,4?-1 and iqm3-2have similar sensitivities to increase or decrease of GA content,and it is speculated that these phenotypes of the tetra mutants are determined by mutations of IQM3 gene.4)Expression analysis of key genes for synthesis and reaction of gibberellin:q PCR quantitative analysis on the reverse-transcription products of RNA extracted from germinating seeds was performed using Actin2 as the internal reference gene.The results showed that GA synthesis key genes GA20ox1 and GA2ox1 were down-regulated in both iqm?1,2,3,4?-1 and iqm3-2.The expressions of GA receptor genes GID1a and GID1b as well as GA response key gene family Dellas?GAI,RGA,RGL1,RGL2,RGL3?,and the F-box protein gene SPY related to Della degradation were up-regulated to different degrees.The expression of these genes in the other three related single mutants was not consistent with the above two mutants.These results indicate that IQM3 may be acted downstream of IQM1,IQM2 and IQM4 in the GA signaling pathway to regulate seed germination,,further confirming that the seed-germination phenotype of iqm?1,2,3,4?-1 may be mainly determined by the mutation of IQM3.5)Flowering phenotype analysis:The flowering time and the number of rosette leaves when flowering were observed and counted under both long-and short-day conditions.The results showed that iqm?1,2,3,4?-1 had delayed flowering and more rosette leaves than Col only under long-day condition.In conclusion,tetra mutants of IQM gene family named as iqm?1,2,3,4?-1 were constructed by CRISPR/Cas9 genome editing technology.Through phenotypic and gene expression analyses,it was found that the phenotype of seed germination for tetra mutants treated with PAC may be determined by the mutation of IQM3,suggesting that IQM3 may be acted downstream of IQM1,IQM2 and IQM4 in the signaling pathway of GA3 to regulat seed germination..