Research On The Molecular Mechanism Of The PTD1 Gene Controlling Rice Plant Height

Plant height is an important agronomic trait for crop architecture and yield.Therefore,elucidation of the molecular mechanism underlying plant height development is significant for plant architecture patterning and crop yield improving.Here,a dominant rice dwarf mutant was studied.It was previously found that the mutant phenotype can be recovered under short-day and high temperature conditions,thus it was designated Photoperiod-Thermo-sensitive Dwarf 1?PTD1?.Later,the PTD1 gene was cloned and sequencing analysis showed that PTD1 carries a point mutation in a gene encoding a non-specific lipid transfer protein?nsLTP?.Most nsLTPs are reported to share a conserved motif called eight-cysteine motif?8 CM?that form four conserved disulfide bonds.However,the 7th and 8th cysteines(Cys¹0202 and Cys¹¹⁶)in the 8 CM are loss in PTD1 due to the frameshift starting from the 101th AA.As a consequence,the intra-disulfide-bond linkages fail to form properly in PTD1.It was previously showed that transgenic plants containing ptd1^C102G/C116G,a modified version of ptd1 with codon conversions from Cys¹⁰²and Cys¹1616 to Gly¹0202 and Gly¹¹⁶,respectively,mimic the dwarf phenotype.This result indicated that loss of the two conserved cysteines is crucial for the dominant gain-of-function in PTD1.In addition,an interacting protein of PTD1,OsUBC25,was found previously by yeast two-hybrid screening,which encodes a putative ubiquitin-conjugating enzyme E2,yet its biological function remains unknown.To further elucidate the molecular mechanism underlying the dominant gain-of-function of PTD1 in controlling plant height,this study focuses mainly on:?1?exploring whether the dominant gain-of-function is universal due to loss of the conserved cysteines in 8 CM by introducing a modified nsLTP gene highly similar to ptd1 to wild type plant with point mutation in the8 CM;?2?screening for interactors of PTD1 and identifying their roles on the dwarf effect;?3?detecting the expression differences of genes involved in Brassinosteroid?BR?pathway in PTD1.The main results are as follows:?1?The dominant function caused by the destruction of conserved cysteines in 8 CM is not universal in other member of nsLTPs.An LTPL17 gene which encodes a protein with high similarity to ptd1 protein was obtained through database screening.A site-directed mutation vector pCAMBIA1300-LTPL17^C111G/C125G,which contains a modified version of LTPL17 with codon conversions from Cys¹1111 and Cys¹2525 to Gly¹1111 and Gly¹2525 in the 8 CM motif,respectively,was constructed and transformed into a wild-type rice variety Zhonghua11?ZH11?.Twenty-five independent transgenic lines were obtained.All transgenic plants showed no abnormal phenotype,indicating that the dominant gain-of-function due to destruction of conserved cysteines in the 8 CM of PTD1 is not universal in other nsLTP member.Thus it is reasonable to speculate that the function of PTD1 probably results from specific interaction between its special conformation and some protein factors.?2?Exploring the role of the interaction of PTD1 with other proteins to the dwarf phenotype.The interaction between a candidate factor OsUBC25 and PTD1/ptd1 was verified by Y2H and Pull-down experiments.Further,the endogenous OsUBC25 gene in wild-type plants was knocked out by CRISPR/Cas9 system.The resluted knockout plants present a dwarf phenotype similar to PTD1,indicating that the dwarf phenotype of PTD1may be due to loss of function of OsUBC25.To further reveal the protein interaction of PTD1,Semi Pull-down method combined with mass spectrometry analysis was performed,and 181 and 51 candidate interactors of ptd1 and PTD1 were identified,respectively.Among them,ArsM1 is a specific factor present only in PTD1 Pull-down products,the homologous gene of which is SMT2 in Arabidopsis,functioning upstream of BR synthesis pathway.It was reported that the smt2 mutant displays dwarf and growth inhibition,suggesting that ArsM1 may involve in the dwarfism of PTD1.A CRISPR/Cas9 knockout vector and an over-expression vector were constructed to study whether the original function of ArsM1 is related to plant height regulation,and the constructs are now undergoing rice genetic transformation.?3?Analyzing the expression of BR pathway related genes in PTD1 mutant.The results of qRT-PCR showed that several genes of BR pathway are expressed abnormally in the stem of PTD1,indicating that PTD1 dwarf may involve BR pathway.