Primary Study On The Relationship Between DTH8 And Hd1 In Regulation Network Of The Rice Photoperiodic Flowering

Heading date,which is affected by both environments factors and internal factors,is an important agronomic trait.Regulation of rice heading date form a complex control networks under various factors.Hdl,which is homolog of CONSTANS(CO)in Arabidopsis,is a crucial regulator of rice photoperiodic flowering pathway.Different to CO,Hdl is a bifunctional gene in rice:Hdl can promote Hd3a expression under SD condition,but it can convert to suppress Ehdl expression under LD condition.DTH8 is also an important gene in rice flowering time regulation.DTH8 that encodes a CCAAT-box binding protein delay rice flowering under LD condition by regulating expression of Ehdl.The mechanism of plants response the environment factors is that the various receptors of mesophyll cell membrane recognize external factors and transduce the external signals into internal signal pathway.The different external factors will get diverse transduction and form different signal pathway.The different signal pathway probably induce a comprehensive effect on plant growth and development by signal cascade amplification,crisscross,overlapping and integration.The base of these signal pathways is protein-protein interaction.We can get mutants as fast as we want using CRISPR/Cas9 genome editing technology,which is a new and developing fast tool in recent years.The successfully application of CRISPR/Cas9 for multiplex genome editing in plants make the research of genetic relationship in rice more fast and convenient.In this paper,we identify the protein-protein interaction of flowering related genes DTH8 and Hdl by A series of biochemical experiments,including Yeast Two-hybrid assay,BiFC,LCI assay and In vitro pull-down assay.Besides,we get many transgenic lines,containing dth8,hd1 and dth8hdl mutants with CRISPR/Cas9 system.With those mutants,we can analyze the genetic relationship of DTH8 and Hdl in rice photoperiodic flowering regulation network.The detailed results are as follows:1.We constructs a series of recombinant vectors:Containing pGBKT7-Hd1,pGBKT7-DTH8,pBADT7-Hdl and pBADT7-DTH8 of GAL4 Yeast two-hybrid system;pB42AD-Hd1 pB42AD-DTH8,pLexA-Hdl and pLexA-DTH8 of LexA Yeast Two-hybrid system;YFPC-Hdl and YFPN-DTH8 of BiFC assay;pET32a-Hdl and pGEX-6p-1-DTH8 of prokaryotic expression vector;PCAMBIA1300-cLUC-DT-H8 and PCAMBIA 1300-Hd1-nLUC of LCI assay;The CRISPR/Cas9 vectors of multi-target sites.2.The yeast two-hybrid assays results of GAL4 and LexA indicate that DTH8 and Hd1 has interaction in yeast.3.YFP fluorescence observation of BiFC in rice protoplasts suggests that YFPC-Hd1 and YFPN-DTH8 can interact in rice protoplast.In addition,the co-localization of the interaction position of Hd1 and DTH8 with nucleus.4.pET32a-Hd1 and pGEX-6p-1-DTH8 is induced by IPTG in Transtta(DE3),which is cultivated by LB with antibiotics,then the fused protein purification with Ni-NTA beeds and GST beeds by affinity purification method.The In vitro pull-down have not a nice result that can’t perfect explain the interaction of GST-DTH8 and Hd1.5.The quantification of LUC activity and imaging with a low-light imaging system demonstrates that DTH8 and Hd1 interaction in tobacco transient expression system.6.The CRISPR/Cas9 vectors of multi-target sites was introduced into Dongjin to obtain transgenic plants by Agrobacterium-mediated transformation.7.We successfully get the hd1,dth8 and hdl dth8 mutants through PCR to amplify the DNA fragments near the target sites of DTH8 and Hd1 with specific primer,and then comparison the sequence results and target gene with DNAMAN software.