Cloning And Functional Identification Of Suppressor Of High Blue Light Insensitive Mutant P2sa1 In Arabidopsis

As a signal,blue light not only participates in regulating the morphogenesis of plants,but also affects the movement of plants,including chloroplast movement,stomatal movement and leaf extension.The phototropism is an important way for plants to guarant the best orientation of plant growth and avoid high light damage.Therefore,it has a great significance to analyze the signal network for further exploring the signal transduction mechanism of plant phototropism.The phototropic response of plant involves multiple photoreceptors,mainly regulated by phototropins and cryptochromes.PHOT1(Phototropin 1)and PHOT2(Phototropin 2)sense blue light and regulate phototropic of hypocotyls in Arabidopsis thaliana in a redundant way.PHOT1 not only regulates phototropic curvature of hypocotyl induced by HBL(high-intensity blue light),but also mediates the response induced by LBL(low-intensity blue light).However,PHOT2 only acts in HBL.In order to research the phototropic mechanism mediated by PHOT2,the gene of P2SA1(Phototropin 2 Signaling Associated 1)was cloned.Our previous researches have proved that P2SA1 as a downstream signaling moleculer only response to HBL,but the specific function of P2SA1 in the blue-light signal transduction mechanism remains unclear.In order to further study the function of P2SA1 in the blue-light signal transduction,the T-DNA inserted mutant of p2sal was used for EMS mutagenesis to screen the suppressors of mutant p2sa1.We finally get a mutant of M120 that have hypocotyl phototropism in response to unilateral HBL and single recessive mutant genes that can be stable genetic.Our previous research suggested that M120 was preliminary mapped on the upper of Arabidopsis thaliana telotrisomic chromosome 2.According to the results of whole genome high-throughput sequencing,we selected AT2G04360,AT2G04740,AT2G05520,AT2G05830,AT2G07170 as candidate mutant genes and ordered related mutant materials.To reduce the cloning region of M120,we expend the number of cloning group and reduce the clone region to 5.57M-5.79M of chromosome 2 by fine mapping.In addition,using Mutmap sequencing technology for backcross sequencing.According to the results of fine map,backcross sequencing and related literature reported,we finally selected AT2G13600,AT2G12935,AT4G29130,AT5G65440,AT5G65490,AT5G63860 as candidate mutant genes and cloned these genes for sequencing verification.The sequencing results indicated their exons all mutated.And then we ordered related candidate mutant materials.We identifed the T-DNA insertion of candidate mutant materials and the results showed that 12 of them are T-DNA insertion homozygous mutant.The phototropic identifed results showed that these 12 homozygous mutants hypocotyl all presented hypocotyl phototropic in response to unilateral HBL.Then we hybridized these mutants with p2sa1 to obtain F1 generation.The F2 generation was screened from the offsprings of F1 selfing generation.Then from the F2 generation identify double mutant.We have identified 9 homozygous double mutants,including 7 homozygous double mutants which did not restore hypocotyl phototropism and the other 2 double mutants restored the hypocotyl phototropism phenotype of p2sa1.In addition,We have constructed 6 T2 generation Cas9 transgenic plants of P2SA1 by the method of gene knockout and 5 T1 generation complemented transgenic plants of M120.In addition,we screened the M2 generation of homozygous T-DNA inserted mutant p2sa1 to obtain others mutant that can restore the phototropic phenotype of p2sa1 in response to unilateral HBL.Through preliminary screened about 20000 M2 generation EMS mutagenesis,3 stable inheritance strains(M2-10,M2-22,M2-43)and 1 hypocotyl short mutants(M2-7)were constructed.Genetic analysis showed that the phenotype of M2-7 was controlled by single recessive gene.We have constructed 2500 map-based cloning samples and mutation site was preliminary mapped on the upper of Arabidopsis thaliana telotrisomic chromosome 2.The results of backcross sequencing showed that the GA(Gibberellin A)receptor GID1 coding gene AT3G05120 has occurred non-synonymous mutation.The sequencing results indicated that the 859th base G was replaced by A,this gene mutation result in 286th glycine was replaced by serine.We hypothesized that the AT3G05120 gene mutations may cause the hypocotyl growth defect of M2-7.In order to verify this speculation,we treated M2-7 with different concentrations of GA3 and GR-24(Strigolactone analog).We found the hypocotyl of M2-7 not elongated when treated with GA3 but elongated when treated with GR-24.This phenomenon indicated that M2-7 is specific in response to GA3.So we ordered T-DNA inserted mutant seeds of M2-7 and constructed complemented carrier of AT3G05120 for retro-complementation to verify their functions.In addition,light can inhibit the hypocotyl elongation.In order to explore whether M2-7 is specific in response to different light,we treated M2-7 with different light and found the hypocotyl elongation of M2-7 all were inhibited.This result proved that the hypocotyl elongation defect of M2-7 is not regulated by influence the signal transduction of light.Combined with the above experimental analysis,we believed that the hypocotyl elongation defect of M2-7 may caused by AT3G05120 gene mutations.