Studies On Transcriptional Regulation Mediated By Phytohormones And Physiological Characteristics Of Rice Chalkiness Formation

Rice chalkiness is an important grain quality trait,which is formed by some loosely packed starch granules and protein bodies with air spaces in endosperm.Chalkiness not only directly affects the appearance quality of rice,but also the cooking quality and nutritional quality of rice.The occurrence of chalkiness greatly limits the improvement of rice quality and its market price.In China,especially in southern China both early season indica rice and japonica rice have the problems of high chalky rice rate and chalkiness degree.Therefore,improving rice quality,reducing chalky rice rate and chalkiness degree are the focus of rice quality improvement in China.Previous studies have shown that chalkiness is a quantitative trait regulated by multiple genes and also affected by environmental factors.Although some progress has been made in the physiological mechanism of chalkiness formation,the molecular mechanism of chalkiness formation is still unclear.In this research,the relationship between rice chalkiness formation and changes in physiological characteristics of rice was analyzed by using Xiangzaoxian 24（X24,high chalkiness）and its origin parents Xiangzaoxian 11（X11,high chalkiness）and Xiangzaoxian 7（X7,low chalkiness）,and the transcriptional regulation of rice chalkiness formation mediated by phytohormones were studied.The results were as follows.1.Dynamics of chalkiness formation showed that endosperm of X7 began to translucent,while the endosperm of X11 and X24 began to emerge opaque at belly of endosperm since20 DAH.Scanning electron microscopy showed that the endosperms of X11 and X24 carried small irregular shaped and loosely packed starch granules with air spaces,while endosperms of X7 were filled with large,regular and tightly packed starch granules at 8 DAH.We also found the photosynthesis rate of X11 and X24 at early grain filling was higher than that of X7.In addition,grain filling rate,fresh weight and water content,amylose content and soluble protein content,ABA,IAA,ZR and GA₄ content,MDA content,SOD and POD activities,mineral content were also differences between high chalkiness and low chalkiness of grains.2.Transcriptome analysis showed that there were 1022,1000 and 1088 DEG_HL at 8DAH,12 DAH and 16 DAH,and these DEG_HL were enriched in many GO functional subclasses and KEGG pathways.Many genes involved in starch,sucrose and protein metabolism were differentially expressed between high and low chalkiness grains at 8 DAH,12 DAH and 16 DAH.At the same time,many genes involved in phytohormonal metabolism and signaling pathway were differentially expressed between high and low chalkiness grains.In addition,analysis of DEG_HL encoding transcription factors and protein kinases showed that several DEG_HL were reported to be involved in phytohormonal metabolism or signal transduction.3.Two candidate genes,hydrolase,alpha/beta fold family protein（Os R498G1018986900.01）and glycosyltransferase（Os R498G0917707800.01）were selected by transcriptome sequencing and the q RT-PCR results,their expression in high chalkiness grains were significantly higher than that in low chalkiness grains,and there were little difference in expression between the two high chalkiness grains.Meanwhile,we found that the promoter of both two candidate genes contain cis-acting elements realted to phytohormones,it is suggested that they play roles mediated by phytohormones.Knockout and overexpression lines of two candidate genes were obtained by constructing knockout and overexpression vectors for rice genetic transformation in X11 and X7.4.X24 was conducted as material with 15 mg/L ABA treatment at 4 DAH and 8 DAH.The results showed that chalkiness of ABA-T（ABA treatment group）was significantly lower than that of CK（control）.At 12 DAH,ABA and ZR contents in ABA-T was significantly higher than that in CK,and GA₄ content in ABA-T was significantly lower than that in CK.Meanwhile,transcriptome analysis of grains of ABA-T and CK showed that at12 DAH and 16 DAH,there were 345 and 605 differentially expressed genes between ABA-T and CK,involved in many GO functional subclasses and KEGG pathways.In starch and sucrose metabolism,the expression of amylase genes were up-regulated and the expression of amylase genes were down-regulated in ABA-T.In addition,many genes involved in phytohormonal metabolism and signaling pathway,and many genes encoding transcription factors were also significant differentially expressed at 12 DAH and 16 DAH between ABA-T and CK.