利用CRISPR/Cas9基因编辑技术构建水稻OsSLR1敲除突变体

GA是种子萌发的促进因子,能解除种子休眠和刺激萌发,GA的信号通路可以通过对DELLA蛋白的去抑制作用来完成的。通过对不同物种的DELLA蛋白功能的研究,发现不同物种的DELLA蛋白的功能具有高度保守性,本实验主要研究水稻中的DELLA蛋白SLR1,利用CRISPR/Cas9基因编辑技术构建OsSLR1基因敲除突变株。首先,通过对OsSLR1的结构和功能域进行分析比对,最终选择3个靶点;然后,利用重组法构建CRISPR/Cas9敲除载体;最后,通过农杆菌介导法,将敲除载体转染成熟的日本晴水稻愈伤,待培育出幼苗,通过PCR和Western Blot进行鉴定。最终筛选得到1株Os SLR1敲除突变株系slr1-1,通过观察突变体萌发表型,发现slr1-1突变体第2天发芽率显著高于日本晴,而且slr1-1突变体的生长速度显著快于日本晴。通过不同浓度PAC对slr1-1突变体萌发表型的影响,发现水稻的DELLA蛋白SLR1敲出的突变体种子萌发不受PAC抑制,表明OsSLR1是通过调控GA信号途径影响水稻种子的萌发,详细的调控机制和互作蛋白等还有待深入的研究。通过对SLR1调控GA信号通路控制水稻的萌发的研究,提高水稻的萌发率,保持优良的水稻种子萌发力,为解决水稻种植上的出芽率不齐等问题提供新的品种。

OsNAC120 balances plant growth and drought tolerance by integrating GA and ABA signaling in rice

The crosstalk between gibberellin(GA)and abscisic acid(ABA)signaling is crucial for balancing plant growth and adaption to environmental stress.Nevertheless,the molecular mechanism of their mutual antagonism still remains to be fully claried.In this study,we found that knockout of the rice NAC(NAM,ATAF1/2,CUC2)tran-scription factor gene OsNAC120 inhibits plant growth but enhances drought tolerance,whereas OsNAC120 overexpression produces the opposite results.Exogenous GA can rescue the semi-dwarf phenotype of osnac120 mutants,and further study showed that OsNAC120 promotes GA biosynthesis by transcriptionally activating the GA biosynthetic genes OsGA20ox1 and OsGA20ox3.The DELLA protein SLENDER RICE1(SLR1)interacts with OsNAC120 and impedes its transactivation ability,and GA treatment can remove the inhi-bition of transactivation activity caused by SLR1.On the other hand,OsNAC120 negatively regulates rice drought tolerance by repressing ABA-induced stomatal closure.Mechanistic investigation revealed that OsNAC120 inhibits ABA biosynthesis via transcriptional repression of the ABA biosynthetic genes OsNCED3 and OsNCED4.Rice OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9(OsSAPK9)physically interacts with OsNAC120 and mediates its phosphorylation,which results in OsNAC120 degradation.ABA treatment ac-celerates OsNAC120 degradation and reduces its transactivation activity.Together,our findings provide evidence that OsNAC120 plays critical roles in balancing GA-mediated growth and ABA-induced drought tolerance in rice.This research will help us to understand the mechanisms underlying the trade-off between plant growth and stress tolerance and to engineer stress-resistant,high-yielding crops.

RID1 sets rice heading date by balancing its binding with SLR1 and SDG722

Rice(Oryza sativa) is a major crop that feeds billions of people, and its yield is strongly influenced by flowering time(heading date). Loss of RICE INDETERMINATE1(RID1) function causes plants not to flower;thus, RID1 is considered a master switch among flowering-related genes. However, it remains unclear whether other proteins function together with RID1 to regulate rice floral transition.Here, we revealed that the chromatin accessibilityand H3 K9 ac, H3 K4 me3, and H3 K36 me3 levels at Heading date 3 a(Hd3 a) and RICE FLOWERING LOCUS T1(RFT1) loci were significantly reduced in rid1 mutants. Notably, RID1 interacted with SET DOMAIN GROUP PROTEIN 722(SDG722), a methyltransferase. We determined that SDG722 affects the global level of H3 K4 me2/3 and H3 K36 me2/3, and promotes flowering primarily through the Early heading date1-Hd3 a/RFT1 pathway. We further established that rice DELLA protein SLENDER RICE1(SLR1) interacted with RID1 to inhibit its transactivation activity, that SLR1 suppresses rice flowering, and that messenger RNA and protein levels of SLR1 gradually decrease with plant growth. Furthermore, SLR1 competed with SDG722 for interaction with RID1. Overall, our results establish that interplay between RID1, SLR1, and SDG722 feeds into rice flowering-time control.

Pyramiding of the dep1-1 and NAL1NJ6 alleles achieves sustainable improvements in nitrogen-use efficiency and grain yield in japonica rice breeding

Rice is one of the most important cereal crops in the world, and a substantial increase in grain yield is necessary for food security. However, high yields of semidwarf modern rice varieties are heavily dependent on the application of mineral nitrogenous fertilizer (Tilman et al., 2002;Sun et al., 2014). Nitrogen (N)-insensitive sponses associated with reduced N-use efficiency (NUE) is a major characteristic of the green revolution varieties (GRVs), in which the growth-inhibiting protein SLENDER RICE1 (SLR1) accumulates (Li et al., 2018). Unfortunately, increasing the level of N fertilizer use to reach the full yield potential of GRVs is subject to diminishing returns, quite apart from its deleterious effect on the environments (Rahn et al., 2009;Liu et al., 2015). Therefore, there is an urgent need to develop new rice GRVs that increase NUE while maintaining their high yields. Recently, several genes (e.g., DEP1, OsNRTl.lB, OsNRT2.3b, ARE1 and GRF4) responsible for improved NUE have been identified in rice (Sun et al.. 2014;Hu et al., 2015;Fan et al., 2016;Wang et al., 2018;Li et al., 2018). More importantly, boosting the activity of the transcription factor GRF4 overcomes the ability of SLR1 to prevent the GRF4-GIF1 interaction, which in turn promotes the coordinated expression of the genes involved in N assimilation and carbon fixation and consequently enhances the NUE of rice GRVs, thereby improving our ability to grow crops sustainably (Li et al., 2018). However, current understanding of the genetic basis for improving NUE remains at the level of identification of a number of quantitative trait loci (QTLs), without any understanding of the nature of the gene products.