The Cloning And Identification Of The Hsp Gene In A Floral Mutant Arabidopsis Thaliana

In this study,a novel flower stem mutant of the Landsberg ecotype,named as a810,was screened from the EMS-mutated Arabidopsis thaliana.The mutant growth morphological characteristics were identified,the mutation gene was predicted and cloned based on the Illumina HiSeq^TMM sequencing.The obtained results as follows.1.The homozygote of a810 was generated for the morphological characteristics observation,we found that the plant showed some stamens infertility,short carpels or three carpels,and some developed stamens and carpels in the carpels?two rounds in a flower organ?in homeotic mutation trait in incomplete floral organ organization in the florets developed early stage.The mutant trait was similar to the kun mutant or miRNA and AGO-related mutations,which exhibited typical floral stem cell regulation characteristics,yet the mutant phenotypes associated with miRNA and AGO were significantly different in that the late inflorescence and floral organ growth were normal,and cloning has ruled out mutations in the KNU genes.Therefore,the mutant may be a new floral stem cell regulatory mutant.To identify the mutant gene in a810 mutant,we constructed an Arabidopsis sample pool for Mut-map sequencing and a sequencing library.The Illumina HiSeq^TMtechnology was used to Mutmap sequence,three candidate genes were selected:hypothetical protein BpHYR1?022900,AT2G41490,AT3G47940;We selected the heat shock protein AT3G47940?AtHSP?gene to carry out gene cloning and biological function research.2.To verify the biological function of AtHSP gene in a810 mutant,the specific primers were designed according to the AtHSP gene sequence of Arabidopsis from NCBI,the AtHSP gene of Arabidopsis Ler ecotype were cloned by homologous recombination,35S::AtHSP constitutive expression vector and AtHSPpro::At HSP specific expression vector was constructed,then were transformed into a810 mutant,the morphological characteristics of flower organs and pods?Carpels?of transgenic plants were checked:the results indicated that a810 mutant trait did not change significantly in the transgenic plants,the complementary effect on the a810 mutant was not achieved in the T₀ generation AtHSP.It was worth noting that the early inflorescence of T_1,T₂ and T₃ transgenic plants showed the traits of the early fruit pods gradually recovered with the increase of the planting algebra,and the three carpels and the carpels developed,the complete floral organ traits decreased from generation to generation,and the short carpels gradually returned to normal,and the phenomenon of ectopic expression of flower organs almost disappeared.3.The elements response for heat shock ABA and MeJA were selected to further analyze its biological functions by analyzing the promoter elements of AtHSP gene,the Arabidopsis Ler was transformed by constructing the GUS gene reporter vector including AtHSP promoter,and the biological function of At HSP was analyzed from the perspective of promoter.The results indicate:?1?By designing 40°C heat shock at different time points,different concentrations of ABA,MeJA combined with heat treatment at 40°C,the germination rate of a810 was significantly lower than the wild type,even the growth of roots and hypocotyls was inhibited and shorter than Ler with increasing heat shock time,the germination rate of a810/Ler and the root length of plant seedlings were significantly or extremely significantly different.The combination of heat shock and ABA significantly inhibited the germination of a810/Ler seeds,and the inhibition of a810 was more obvious,the germination rates of the two were significantly different,when the ABA concentration was 0.05?mol/L,the germination rate of a810/Ler seeds without heat shock treatment was significantly higher than that the without ABA and higher concentration of ABA.At the same time,with the increase of ABA concentration,the growth of seedlings and roots was gradually inhibited,under the same growth conditions,the seedling growth and root length of a810 were weak or lower than Ler.When the concentration of MeJA improved,the germination rate of a810/Ler seeds increased,under the three temperature conditions set by the experiment,the same concentration of MeJA treatment,the germination rate of both at 28°C is significantly higher than the treatment of 23°C and 40°C;the germination rate of a810/Ler treated with heat shock at 40°C increased slightly with the increase of MeJA concentration,but the germination rate was much lower than 28°C because of the heat shock.At the same time,the lower concentration of MeJA promoted the growth of a810/Ler seedling roots under three temperature conditions and promoted at 28°C,however,after heat shock treatment at 40°C,the control and different concentrations of MeJA there was a significant difference in root length between the a810 and Ler seedlings.?2?We observed the potassium iodide staining of mature pollen of a810/Ler in early inflorescence,the fertility rate of Ler pollen was 95.05%,while the pollen fertility rate of a810 mutant was only 38%.It indicated that the infertility of a810 mutant may be related to the pollen fertility,It is speculated that the mutation of AtHSP gene in a810 mutant may cause some pollen fertility to decreased,which may affected the normal fertilization of ovule and cause seed abortion.?3?To verify the expression of AtHSP gene in Arabidopsis thaliana,we cloned the specific promoter sequences of AtHSP gene with length of 2782 bp,2052 bp and 1643 bp,then we constructed At HSPpro::GUS specific reporter vector and transformed Ler plant.GUS staining of three transgenic plants revealed that there were weak expressions in the 4th and 5th fruit pods?seed endosperm?and mature pollen of mature plants.This result is basically consistent with our predicted gene expression profile.