Map-based Cloning And Functional Analysis Of SGD2,A Small Grain And Dwarf Gene In Rice(Oryza Sativa L.)

Plant height is a significant agronomic trait in rice,which determines the lodging resistance of rice,ideal plant architecture and the yield of rice.Identification and utilization of new dwarf resources,will help accelerate the process of high-yield rice breeding and provide theoretical base for molecular design breeding to improve rice plant architecture.This paper used a small grain and dwarf rice mutant as the research material,which named small grain and dwarf 2?sgd2?.SGD2,a key factor affecting the growth and development of rice,was isolated by map-based cloning method.Further researches revealed the molecular mechanism of SGD2.The results are summarized as follows:1.Compared with the wild-type Kitaake,the mutant sgd2 showed obvious growth retardation phenotype at the vegetative growth stage.The seed germination rate was low,the seedling plant was short,the root length became short,and the growth rate was relatively slow.After heading stage,the plant height was about 18.7 cm,the tiller number was decreased,the internode and panicle were all shortened,and the seed setting rate was only about 4%.sgd2 seeds showed a phenotype of small round grains.The grain length and width of sgd2 seeds decreased significantly,and the 1000-seed weight was only about 18.2 g,which was 34.5%lower than the 27.8 g of Kitaake.2.Confocal laser microscopy showed that the SAM area of sgd2 was significantly smaller than that of Kitaake.Uppermost internode cell observation showed that mature plants sgd2 longitudinal cell length less than Kitaake.Meanwhile,a transverse cell area have also been dropped significantly,we speculated sgd2 dwarf is mainly caused by the decrease in the length of internode cells.Scanning electron microscopy of the outer epidermal cells of the mature seeds lemma showed that the longitudinal cells of sgd2 were shortened,which was speculated to be the direct cause of the decreased grain size.3.By means of genetic analysis,we confirmed that SGD2 was a recessive gene in sgd2 mutant.F₁of the offspring of sgd2×IRAT129 all showed normal plant height phenotype.The genetic population of F₂ offspring was used to map the small grain and dwarf gene,which located at the long arm of rice chromosome 1 about 83.8 Kb,and the genome sequencing found that the Os01G0643600 gene got 9-bp deletion mutantion.Gene complementary and knock-out assays indicated that,Os01G0643600 gene was a positive regulation gene of rice plant height and grain size,named SGD2.Further study showed SGD2 was annotated as HOX3,a member of the HD-Zip II family transcription factor.4.RT-PCR and GUS staining showed that SGD2 was a compositional expression gene,which was expressed in young roots,seedlings and stems of rice,and was highly expressed in young panicles before flowering.Based on the transient expression of rice protoplast and the observation of young roots from SGD2-GFP rice transgene plants,it was found that SGD2 was mainly located in the nucleus.The results of yeast and luciferase assay showed that SGD2 was a transcriptional inhibitor and did not have transcriptional activation function.5.Plant physiology experiments showed that sgd2 could respond to the exogenous GA₃ treatment at seedling stage,and the length of the second leaf sheath could be restored to Kitaake level.The spraying treatment of GA₃ with different concentration gradient before heading stage showed that the response degree of sgd2 and Kitaake was almost the same,and there was no significant difference in plant height elongation ratio.GA20ox1 gene was overexpressed under the sgd2 background,and the transgenic plants presented high stem phenotype.HPLC experiments showed that the content of endogenous GA₁in sgd2 plants decreased by 2/3 compared with Kitaake,suggesting that the gibberellin synthesis pathway of sgd2 was affected.6.The yeast two-hybrid experiments proved that SGD2 could interact with its homologous proteins HOX1 and HOX2,and all three proteins could target to D18?GA3ox2?gene promoter.The sgd2/d18double mutant showed a cumulative effect,the plant height of which was lower than that of sgd2 or d18,suggesting that sgd2 and d18 may be in the same regulatory pathway.