| Chloroplast chaperone CPN60(Chaperonin 60)belongs to the molecular chaperone family,which can help to fold and assemble a variety of substrates.It mainly divides into two groups,type? and type?.CPN60 belongs to type I molecular chaperone.In Arabidopsis thaliana,CPN60?1,CPN60?2,CPN60?1,CPN60?2,CPN60?3,CPN60?4 are the protein subunits that make up the structure of CPN60,and three co-chaperones,CPN10-1,CPN10-2 and CPN20.At present,although different mutants have been reported on the six protein subunits of CPN60,the relationship between the six subunits is still unclear.There are few reports on CPN10 and CPN20 co-chaperones in chloroplasts.Therefore,the focus of this study is to create CPN10 and CPN20 mutants by using the T-DNA mutant identification method,at the same time,Through the acquisition of transgenic plants way to explore the links of six subunits of the CPN60,The results obtained in this study are as follows:(1)Chloroplast co-chaperone CPN10 has two proteins,CPN10-1 and CPN10-2.In this study,two T-DNA insertion mutants of CPN10-1 were ordered and named cpn10-1-a and cpn10-1-b.Two T-DNA insertion mutants of CPN10-2 were ordered and named cpn10-2-a and cpn10-2-b.cpn10-1-a,cpn10-1-b,cpn10-2-a,cpn10-2-b homozygous mutants were identified,and their phenotypes were similar to those of wild-type Arabidopsis thaliana.Because the amino acids of CPN10-1 and CPN10-2 share 84% similarity,cpn10-1-b x cpn10-2-b were hybridized to obtain a homozygous double mutant of cpn10-1-b x cpn10-2-b.Compared with the wild-type plant,the homozygous double mutant showed a yellowing dwarf phenotype,indicating that CPN10-1 and CPN10-2 had similar functions and redundancy.(2)In this study,two T-DNA insertion mutants of CPN20 were ordered,named cpn20-a and cpn20-b.A homozygous mutant of cpn20-a was identified,and its T-DNA was inserted in the UTR region of the gene,which was a weakly expressed mutant.Compared with the wild-type Arabidopsis plant phenotype,the homozygous mutant cpn20-a exhibited a yellow-stunted phenotype.heterozygous mutants of cpn20-b were obtained,but homozygous mutants of cpn20-b were never obtained.The progeny of cpn20-b heterozygous mutant was identified again,and the results showed that the genotype ratio of the progeny of cpn20-b heterozygous mutant was AA: aa = 1:1,and there were no homozygous mutant plants with aa genotype.(3)the progeny of cpn20-b heterozygous mutant appeared two genotypes and the ratio was AA:aa=1:1 to conduct a preliminary investigation.There are two possibilities for the occurrence of the phenomenon: the first is that the recessive gene in the male gamete is lethal;the second is death from a recessive gene in the female gamete.In this study,Alexander dye was used to observe the pollen,and it was found that the pollen of cpn20-b heterozygous mutants was normal,indicating that the male gamete recessive gene of the mutants was normal.Then,the observation and statistical analysis of cpn20-b heterozygous mutant silique showed that some seeds could not develop and mature,indicating that the recessive genes in the female gametes of cpn20-b heterozygous mutant had lethal phenomenon.Meanwhile,cpn20-b mutant was proved to be a lethal female gamete mutant by the method of positive and reverse crossing and the method of obtaining complementary plants of the mutants.(4)When the top of the substrate binding region of CPN60?1 after the loss of 29 amino acids,the plants developed a yellow-dwarf phenotype,suggesting that these 29 amino acids played an important role in CPN60?1.due to the high homology among the six protein subunits of CPN60.these 29 amino acids also play an important role in the other five protein subunits? In this study,transgenic plants with CPN60?1 truncated mutant,CPN60?2 truncated mutant,CPN60?3 truncated mutant and CPN60?4 truncated mutant were obtained.Compared with wild-type plants,these transgenic plants showed no obvious phenotypic changes.These results indicate that the six protein subunits of CPN60 still play different roles in function. |
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