Functional Analysis Of C-terminal Domain Of DEP1 Protein Of Gγ Subunit In Rice

Grain size not only determines the grain weight,but also affects the appearance quality of rice.Studying grain size has important roles for improving rice yield and appearance quality.Studying the mechanism of grain size regulation will provide important genetic resources and theoretical basis for cultivating high-yield and high-quality rice varieties.So far,five regulatory pathways have been found to be involved in the regulation of grain size,including the G protein signaling pathway.In the G protein signal pathway,GS3 inhibits the signal transduction of DEP1 by competitively binding to RGB1.However,both GS3 and DEP1 are atypical G protein γ Subunits,what is the basis of variation of functional differentiation between them,and the understanding of the signal pathway downstream of G protein is still unknown.On the basis of previous work,this study utilized the CRISPR/Cas9 system to detect the deletion of different segments of the C terminal domain of DEP1,in order to identify the functional elements that determine the positive regulatory effect of DEP1 and verify whether they can determine the functional differentiation between GS3 and DEP1;The yeast two hybrid system and Co-lmmunoprecipitation technology were used to screen the interacting proteins of DEP1,so as to further understand the G protein signaling pathway.The results are as follows:1.Two systems were constructed to perform targeted deletions on the C-terminal domain of DEP1 and mutants of the deletion domains Ⅰ+Ⅱ,L,and Ⅲ were generated in ZH11 background.The mutants lack Ⅰ plus Ⅱ or L sequences,resulting in shorter grains.However,the mutant lacks the Ⅰ,Ⅱ,and L sequences,resulting in much shorter grains compared to the individual deletions of I plus II or L.The repeated sequences Ⅰ+Ⅱ and L play an important role in the positive regulation of DEP1.The absence of domain Ⅲ and subsequent sequence deletions did not affect the positive regulatory function of DEP1,as the mutant did not decrease the grain size.2.An overexpression vector DEP1DL containing DEP1 lacking the L domain was constructed and transformed into ZH11.Co-segregation analysis of two independent lines show that overexpression of DEP1DL could still increase grain size.qPCR was used to detect the expression levels of exogenous DEP1DL,as well as endogenous DEP1 and GS3 in 1 cm young panicle of two DEP1DL overexpression lines.The phenotype of larger seeds of transgenic plants was caused by the overexpression of DEP1DL,rather than affecting the expression levels of endogenous DEP1 and GS3.3.Different domains of DEP1 were used to generate bait vectors of Y2H system.Selfactivation detection revealed that the L domain exhibited self-activation activity.A cDNA library of panicle tissue was screened and six candidate genes were obtained.Two DEP1 interacting proteins were finally conformed by yeast two hybrid and Luciferase complementation assay.4.The overexpression plants of DEP1 fused with 3xFlag tag in the N terminal were constructed,and the interacting proteins of DEP1 were screened by protein immunecoprecipitation.A total of 426 differential proteins were obtained,of which 166 were significantly higher in the overexpression plants than in the control ZH11 sample.GO analysis of these 166 proteins showed that DEP1 is associated with the cell plasma membrane and participates in ion channels.The candidate genes were verified by yeast two hybrid and Luciferase complementation assay,and two DEP1 interacting proteins were finally identified.