| Chlorophyll is an important pigment for photosynthesis in plants.The pigment is responsible for the conversion of light into chemical energy via photoreaction in chloroplast,and the chlorophyll content affects photosynthesis.The biosynthesis of chlorophyll is catalyzed by a series of enzymatic reactions.Among them,chlorophyll synthase(CHLG)catalyzes the esterification of chlorophyllide(a and b),the last step of chlorophyll biosynthesis.In this study,Medicago truncatula,a model legume plant,was used to explore the effect of MtCHLG on chlorophyll synthesis by analyzing its expression pattern and constructing MtCHLG knockout vector using CRISPR/Cas9.The main results are as follows:The cloned CDS of MtCHLG was 1,137 bp,encoding 378 amino acids,and the putative MtCHLG belongs to the Ubi A superfamily.Based on bioinformatic analysis,the secondary structure of MtCHLG consists of?-helix(41.27%)and random coils(40.74%).MtCHLG has five transmembrane helices parallelled to one another to form a barrel-like shape.MtCHLG had a high sequence identity with legume CHLG,including Ms CHLG(99.2%)and Gm CHLG(93.9%)from Medicago sativa and Glycine max,respectively.Although CHLGs from 17 select species shared common conserved domains with relatively uniform gene compositio,CHLGs were divided phylogenetically into distinct groups.It seems that several terrestrial plants possess a couple more CHLGs and the length of introns increases significantly.When transiently expressed in tobacco,the fluorescence of the MtCHLG-GFP recombinant protein was predominantly detected in chloroplast of the epidermal cells.MtCHLG was differentially expressed in the tested tissues including both underground and aerial organs with the highest detected in leaves.Analysis of the predicted promoter(2 kb)of MtCHLG revealed multiple cis-acting elements,such as light response,ABA response,Me JA response and drought response.Experimental analysis showed that the expression of MtCHLG was induced by light while inhibited by darkness(<48 h).Under ABA(100?mol·L-1)treatment of 2 h,MtCHLG decreased to about 0.26-fold of the control(P<0.05).PEG(5%)treatment of 24 h repressed the expression of MtCHLG to about 0.42-fold of the control(P<0.05).MtCHLG is downstream of Mt CAO in chlorophyll biosynthesis pathway,and its expression level is effected by the loss of Mt CAO function.Hence,the transcriptional level of MtCHLG is affected by multiple factors.Three MtCHLG knockout vectors were constructed by Golden Gate cloning using CRISPR/Cas9 to target four exons of MtCHLG.The MtCHLG CRISPR/Cas9 constructs,together with the control,were transformed into Medicago truncatula through Agrobacterium-mediated transformation.For the three constructs,52,60 and 98 transgenic positive plants have been obtained with no obvious morphological abnormality.Genotyping the target sites of the positive plants revealed the same sequence to that of the wild type,indicating no editing of the target sites in the transgenic plants.Optimization of CRISPR/Cas9 vector suitable for Medicago is needed. |
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