Fine Mapping Of Rice Grain Number-regulating Gene GNP4 And The Mechanism Of SK41-IAA10-ARF17 Regulating Rice Grain Size

Rice is one of the most important food crops in the world.Increasing the yield of rice per unit area is the key to solving the problem of food security.The number of grains per ear and grain size,as the components of rice yield,have a direct and important impact on rice yield.However,the number of grains per ear and grain size are agronomic traits that are controlled by multiple genes and are greatly affected by the environment.There are few cloned genes and their genetic mechanism and molecular basis are not very clear.Therefore,cloning more new genes related to grain number per panicle and grain size and analyzing the molecular mechanism network of their regulation is of great significance for future high-yield rice breeding.Part 1.Fine mapping of the grain number-regulating gene GNP4 in riceIn this study,the indica rice variety Hu Dasui(large spike phenotype)was used as the donor parent,and the indica rice variety Z104(small spike phenotype)was used as the recurrent parent,and a near-isogenic line was constructed by crossing and continuous backcrossing for the gene GNP4 that regulates grain number per spike.Perform fine positioning.The main findings are as follows:1.Comparing the agronomic characters of two indica parents,Hu Dasui and Z104,found that Hu Dasui had higher plant height,thicker stem,wider leaves and more grains per ear than Z104.Compared with Z104,the ear length of Hu Dasui was significantly reduced,and the primary branch,secondary branch,number of grains per ear and yield per plant were significantly increased.In terms of grain shape traits,compared with Z104,Hu Dasui significantly decreased in grain length,grain width and 1000-grain weight.2.Next,we took 30 recessive spikelets and 30 dominant large spikelets in the BC5F2 population for RICE6 K chip detection,and determined the initial location interval to be about 2.9MB between chromosome 4 RM5503～RM3648 between the location.3.The fine-mapping large population consisted of 4401 strains of BC5F2 and BC5F3.Fine mapping of GNP4 using 13 SSR and INDEL markers.Finally,7 decisively important exchange individuals were obtained.GNP4 was located in the 22.3Kb interval between SNP4～INDEL right.And important exchange single plant is planted for progeny verification.4.The mapping interval contains three genes,LOC＿Os04g52479,LOC＿Os04g52500,and LOC＿Os04g52504.Sequencing comparison found that: LOC＿Os04g52504 has no difference between the two parents,LOC＿Os04g52500 has 7 base substitutions and 2 base deletions in the intron region,and LOC＿Os04g52479 has a total of three base substitutions in the coding region.Arginine(R)on exon is mutated to histidine(H),alanine(A)of the fifth exon is changed to valine(V),and valine(V)is changed to iso Leucine(I).Therefore,LOC＿Os04g52479 was locked as the gene GNP4 that controls the number of grains per ear.5.In addition,we also found that there is indica-japonica differentiation at the natural mutation site of GNP4.This rule was found by sequencing 132 conventional rice varieties in our laboratory: GNP4 in indica varieties is generally GNP4(G)type on the third exon.(CGT),japonica varieties are generally GNP4(A)type(CAT).This finding is unprecedented and can be used as a new indica-japonica differentiation site to study.Part 2.Mechanism of SK41-IAA10-ARF17 regulating rice grain sizeA QTL that controls rice grain length and grain weight,TGW3,was cloned in the previous work in our laboratory.This gene encodes a GSK3(glycogen synthase-like kinase)family protein member,Os GSK5/SK41,which negatively regulates rice grain size.In the process of studying the molecular mechanism of TGW3 regulating rice grain size,we determined the interaction between SK41 and IAA10.The study found that members of the IAA family can form heterodimers with ARF to play a role.The yeast two-hybrid experiment verified the nine transcriptional activators of the ARF family,and found that only ARF6,ARF11,ARF12,ARF17,ARF21,and ARF25 can interact with IAA10.do.Because ARF17 has been reported less,we mainly studied the molecular mechanism of SK41--IAA10--ARF17 regulating rice grain size.The main results are as follows:We demonstrated the interaction between IAA10 and ARF17 by yeast two-hybrid assay and bimolecular fluorescence complementation assay(BIFC).Subcellular localization experiments showed that ARF17 was localized to the nucleus.Next,two knockout types of ARF17 in the ZH11 background were obtained using CRISPR/Cas 9technology.One is arf17-1 with 6bp deletion and the other is arf17-2 with 42 bp deletion.We found that the two arf knockout mutants have significantly reduced grain length and grain width compared with wild-type ZH11.In addition,the plant height of arf17-1 and arf17-2 mutants was significantly reduced,the number of tillers was also greatly reduced,and the yield per plant decreased.Changes in kernel size resulted in a significant reduction in 1000-grain weight of mutant plants.This study found that the phenotype of arf17 knockout was similar to that of IAA10 overexpression.These results suggest that IAA10-ARF17 may be in the same pathway that regulates grain size in rice.