| Maize is an important cereal crop that is being used as food and feed worldwide.There are various genes and genetic pathways which are responsible for maintaining the normal growth and development in all stages from germination to maturity.Therefore,analyzing the key genes in maize development has been one of the important tasks in the field of maize scientific research.In this study,we used a mutant which is seedling lethality named as Maize Seedling Lethality1?MSL1?to carry out the phenotypic analysis?Morphology to Cytology?,and obtained the candidate gene for MSL1 by gene mapping.This candidate gene encodes a?-ketoacyl-CoA synthase?KCS?,which catalyze the very long chain fatty acids?VLCFAs?biosynthesis in plants.The biological function of the gene was explored by the analysis of wax content in leaf epidermis of mutant and RNA-seq transcriptome sequencing.The main results of this study are as follows:1. The phenotype observation of msl1.The mutant of msl1 germinated normally,but stopped growth after 2 weeks,which resulted in seedling lethality.At the same time,the msl1 cannot grow second leaf.The development of mesophyll cell?MC?,wreath structure?KA?,vascular bundle?VB?,mechanical tissue?MT?and other cytological structures were found abnormal in the first leaf of msl1 compared with the wild type?WT?.No significant differences were observed in the morphology and size of the shoot apical meristem?SAM?between msl1 and WT.This indicates that the phenomenon of seedling lethality is not caused by SAM development,but related to the abnormal development of leaves.2. Fine mapping of MSL1.Based on the classical genetic analysis of F2:3 population constructed by self crossing of F2 generation hybrid population obtained from F1inbreeding?A636ŚW23?,it was found that the seedling lethal trait was controlled by a single recessive gene??c2=0.0632?.The gene was initially located within the 4.13Mb region from 229.95Mb to 234.08Mb on chromosome 3 of maize genome by Bulked Segregant RNA-Seq?BSR-seq?.At the same time,kompetitive allele specific PCR?KASP?markers were designed,and the F2:3population were used to narrow the fine mapping region to 570 kb.It contains 11 genes referring to B73 refgen?4 genome.It was found that only Zm00001d044579 gene in mutant contained a missense mutation in the conserved domain sequence which changed the 318th amino acid of the gene from tryptophan?Ser,S?to aspartic acid?Asp,D?.This gene encodes KCS,which catalyze to elongate VLCFAs in plants,and with high similarity sequence to two KCS genes ONION1?Os03g0181500?and ONION2?Os10g0416200?in rice,it was reported that the onion1 and onion2 mutant also caused seedling lethality?Ito et al 2011;Tsuda et al 2013?,and phenotype is also consistent with msl1,so Zm00001d044579 was found to be the candidate gene for MSL1.3. Analysis of wax content in msl1 leaf epidermis.Scanning electron microscopy?SEM?showed that the wax crystals on the surface of msl1 leaves were significantly less than WT,and the over 30-carbon chains length of wax content such as alcohols and alkanes were significantly decreased.It was speculated that MSL1 candidate genes mainly elongate the VLCFAs after those with 30-carbon chains.It also further proved that Zm00001d044579 gene was the candidate gene for MSL1.4. Transcriptome data analysis of msl1.The genes related to wax synthesis pathway in maize were screened for differential expression.The results showed that there were 34differentially expressed genes.Among which,the number of differentially expressed genes at the most was in VLCFAs biosynthesis pathway.There were 11 DEGs in very long chain fatty acid biosynthesis I pathway,which including 6 KCS genes.Combined with the function of the candidate gene of MSL1,it was preliminarily speculated that this phenomenon may be caused by the lack of KCS function in msl1,which produced a feedback regulation or stress protection reaction mechanism in the VLCFAs biosynthesis pathway,then resulting in part of differential expression genes in the VLCFAs biosynthesis pathway. |
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