【作者】 刘琳；

【导师】 付爱根；

【作者基本信息】 西北大学 ， 工程硕士（专业学位）， 2020， 硕士

【摘要】 肽基脯氨酰顺反异构酶(Peptidyl Prolyl cis/trans Isomerase,PPIase)存在于各种生物体中,它的主要功能是通过催化多肽链中X-Pro肽键从顺式向反式转化,加速蛋白质的折叠,是蛋白质折叠的限速酶。植物的叶绿体中有很多PPIase,我们对它们的功能知之甚少。本研究主要用CRISPR/Cas9基因编辑技术和T-DNA突变体筛选方法创建拟南芥叶绿体部分PPIase的缺失突变体,然后对它们生长表型及其在光合作用中的生理功能进行分析,增加对叶绿体PPIase和光合作用的认识。拟南芥叶绿体基质中共有3个PPIases蛋白,分别是CYP20-3、TIG和PIN3。本研究通过鉴定T-DNA插入突变体,得到了缺失突变体cyp20-3(SALK＿001615)、tig(SALK＿037730)和pin3(GK＿068C12),然后通过杂交得到了cyp20-3×tig双重突变体。在表型分析实验中,发现单基因突变体和cyp20-3×tig双重突变体的表型与野生型相比在生长发育方面没有明显的差异,我们推测基质PPIase蛋白可能与其它蛋白的功能存在冗余现象。利用BN-PAGE实验对单基因突变体和cyp20-3×tig双重突变体植物的光合复合体分析,发现基质PPIase对PSII超级复合体的影响相同,它们的缺失均不同程度地导致了PSII超级复合体减少。有意思的是,cyp20-3×tig双重突变体的BN-PAGE带型与cyp20-3相似,却与tig不同,我们初步判定TIG功能的发挥对CYP20-3有一定的依赖。拟南芥类囊体腔中共有16个PPIases蛋白,我们主要对目前没有研究报道的7个类囊体腔PPIase缺失突变体进行了突变体的创建和表型分析。通过鉴定T-DNA插入突变体,得到了缺失突变体fkbp16-4(GABI＿905D10)。利用CRISPR/Cas9基因编辑技术,得到了两个fkbp13纯合敲除突变体株系(fkbp13-38#和fkbp13-43#),一个fkbp17-1纯合敲除突变体株系(fkbp17-1),一个fkbp18纯合敲除突变体株系(fkbp18),fkbp16-1、fkbp19、fkbp16-3还没有得到纯合敲除突变体。通过对已有突变体的生长发育表型分析,发现fkbp16-4比野生型晚抽薹约1.5天,从这个结果可以初步推测FKBP16-4可能参与调控拟南芥开花过程的生理活动;fkbp13-38#、fkbp13-43#、fkbp17-1和fkbp18的表型与野生型相比在生长发育方面没有明显差异,目前不能确定它们的功能,需要更深入的研究分析。本研究致力于拟南芥叶绿体PPIase突变体材料的创建和筛选,并对筛选到的突变体进行初步表型分析,为后续的实验提供了材料上的方便。在我们的分析中,发现部分PPIase的突变对植物造成了比较大的影响,说明其在植物的生命过程中承担着重要的作用,这为叶绿体PPIase更深入的研究指明了方向。更多还原

【Abstract】 Peptidyl prolyl cis/trans isomerase（PPIase）exist in various organisms.Its principal function is to participate in protein folding by catalysing the cis-trans isomerisation of the X-Pro peptide bond in polypeptide chains.And it is the rate-limiting enzyme for protein folding.The chloroplast of plants have a number of PPIases.Nevertheless,their physiological function is poorly understood.To increase understanding for chloroplast PPIase,we create null mutants of many uncharacterized chloroplast PPIases in Arabidopsis thaliana by CRISPR/Cas9 gene editing technology and T-DNA mutant screening method in this study,then we analyzed the growth phenotype of these mutants and their functions in photosynthesis.There are three PPIases proteins in the chloroplast stroma of Arabidopsis thaliana,which are CYP20-3,TIG and PIN3.In this study,cyp20-3（SALK₀01615）,tig（SALK₀37730）,and pin3（GK₀68C12）were obtained by identifying T-DNA insertional mutants,then we had obtained cyp20-3×tig homozygous double mutant through hybridization.Phenotypic analysis experiments showed that there was no significant difference in growth and development between the mutants（cyp20-3,tig,pin3,cyp20-3×tig）and the wild type,we speculated that the stroma PPIase proteins may be redundant with other proteins.It was found that the stroma PPIases have the same effect on PSII supercomplexes in the BN-PAGE experiments and their deletion resulted in reduction of the PSII supercomplexes for varying degree.Interestingly,the BN-PAGE band of cyp20-3×tig is similar to cyp20-3,but different from tig,we propose that the function of TIG is dependent on CYP20-3.There are sixteen PPIases in the thylakoid lumen of Arabidopsis thaliana.We mainly carried out the creation and preliminary phenotypic analysis of seven PPIases which have not been reported.One T-DNA insertional mutant for FKBP16-4 were obtained,that is fkbp16-4（GABI₉05D10）;We had obtained four homozygous knockout mutants by CRISPR/Cas9,they are two different fkbp13 lines（fkbp13-38# and fkbp13-43#）,one fkbp17-1 line（fkbp17-1） and one fkbp18 line（fkbp18）.The homozygous knockout mutants of fkbp16-1,fkbp19 and fkbp16-3 have not yet obtained.In the phenotypic analysis experiment for these mutants,we found that fkbp16-4 was later bolting about 1.5 days than the wild type,it can be preliminarily determined that FKBP16-4 is involved in regulating the flowering of Arabidopsis thaliana from this result;In the growth and development,the phenotypes of fkbp13-38#,fkbp13-43#,fkbp17-1,fkbp18 were not significantly different from the wild type,we need deeper research and analysis to confirm their functions.In this study,we are committed to the creation and screening of chloroplast PPIase mutants in Arabidopsis thaliana,which provides materials for subsequent experiments.We also performed the preliminary phenotypic analysis of these mutants,it was found that the deletion of some PPIase affects plants,so they maybe play an important role in the plant’s life process.Our study points the way for further research on chloroplast PPIase.更多还原