The Role Of PLA3 Gene In Gibberellin Regulating Seed Germination Of Rice

Seed germination is a critical period in plant individual development and plays a decisive role in the reproduction and survival of plant offspring.Seed germination refers to a series of orderly physiological and morphological processes in which seeds start from imbibing.Plant hormones play an extremely important role in this process,such as gibberellins（GAs）to promote the germination of rice（Oryza sativa L.）seeds.PLASTOCHRON（PLA）is a regulation gene for the interval of rice leaf primordium formation.Early studies found that the pla3 mutant has a larger embryonic and germinated phenotype than the wild type,and pla3 seeds are easy to germinate on the female parent（ear sprouting）.PLASTOCHRON3（PLA3）gene may encode glutamate carboxypeptidase.In order to further explore the relevant mechanism of PLA3regulating rice seed germination,this article uses CRISPR-Cas9 gene editing technology to obtain a PLA3 mutant-pla3 with‘Nipponbare’as the background,and constructed pan580-Os PLA3-GFP fusion protein expression vector to investigate PLA3 protein subcellular localization.At the same time,the liquid-mass spectrometry（LC-MS/MS）method was used to analyze the GA content in the embryos of rice seeds at the early stage of germination（12、30 h）,combined with bioinformatics analysis and transcriptome sequencing to screen GA signal pathway differential genes,preliminary The role of PLA3 gene in regulating rice seed germination by GA was proved.The main results are as follows:1.PLA3 bioinformatics analysis:Through multi-species homology comparison and evolution analysis of PLA3 protein,rice PLA3 protein has high homology with Arabidopsis AMP1（Altered meristem program）and maize VP8（Viviparous 8）;meanwhile,PLA3 The spatiotemporal expression and expression of different hormones were analyzed,and it was found that PLA3 was highly expressed in early embryos and was induced by gibberellin（GA）and abscisic acid（ABA）.PLA3 protein signal peptide,transmembrane region and phosphorylation analysis results show that PLA3 protein has no signal peptide sequence,no transmembrane region structure,is a non-transmembrane protein,and PLA3 protein has different degrees of phosphorylation sites in different positions.2.PLA3 protein subcellular localization:First,Wo LF PSORT was used to predict the subcellular localization of PLA3 protein,and it was found that the protein was more likely to be located in the cytoplasm（64.2%）.In order to further clarify the subcellular localization of PLA3 protein,this paper constructed pan580-Os PLA3-GFP fusion protein expression vector,using PEG-mediated transfer into rice protoplasts,and observed the expression of the fusion protein under the laser confocal microscope.The results showed that the fusion expression protein of the target gene PLA3 and GFP could clearly observe the fluorescence in the endoplasmic reticulum region in the green fluorescent GFP,bright field fluorescence and the superimposed channel of the two,and finally determined that the PLA3 protein was located at Endoplasmic reticulum.3.The relationship between PLA3 and GA and its effect on rice seed germination:Compared with embryos of wild-type dry seeds,pla3 showed a phenotype of large embryos and low endosperm ratio,and pla3 seeds were easy to germinate.The results of seed germination experiments further show that pla3 has the characteristics of earlier germination time and faster germination speed than the wild type.The content of GAs and ABA in the embryos of wild-type and pla3 seeds at the early stage of germination（12,30 h）were determined by liquid-mass spectrometry（LC-MS/MS）.The results showed that the content of GA₄ and GA₉ in wild-type"Nipponbare"At the two stages（12 and 30 h）,the content was higher than that of mutant pla3,but the ABA content of pla3 was significantly lower than that of wild type.It can be seen that PLA3 gene can coordinate with GA₄ to regulate the early germination of rice seeds.The results of transcriptome sequencing are as follows:GA signal pathway genes such as GID1 were significantly up-regulated at the 30th hour after germination of pla3;while the negative regulatory factors SLR1 and SPY did not change significantly.It is speculated that PLA3 gene inhibits the expression of GID1,so that the germination speed of pla3 is significantly faster than that of wild type.The 6 genes in GA signal and metabolic pathway were selected for q PCR verification and analysis,and it was found that the expression trends of transcriptome sequencing and q PCR transcripts were consistent.The above research results indicate that PLA3 gene affects seed germination and participates in the process of GA regulating seed germination.