叶片黄化是植物衰老的重要标志之一。逆境会加速植物叶片中叶绿素和蛋白质降解，进而导致叶片早衰。逆境诱导的叶片早衰不仅降低了草坪质量，也影响了饲草品质。因此在草坪草和牧草两用型多年生黑麦草（Lolium perenne L.）叶片中阻断或抑制叶绿素的降解，理论上可以获得滞绿型和高饲草品质的转基因种质资源。深入研究多年生黑麦草叶绿素降解通路及其调控机制，可以为多年生黑麦草转基因育种提供理论基础。本研究以多年生黑麦草为材料，克隆得到了叶绿素降解的核心调控基因STAY-GREEN (LpSGR)，并对其功能和调控叶绿素降解机理进行了系统分析。 根据已知的拟南芥和水稻滞绿基因信息，采用RACE-PCR技术在多年生黑麦草中克隆得到了滞绿基因LpSGR（NCBI基因数据库登录号为AQM55942）。LpSGR基因编码区由864个碱基组成，其编码了287个氨基酸，且具有典型的SGR功能域。LpSGR基因组序列与单子叶植物（例如水稻、二穗短柄草、高粱和玉米）的SGR基因组结构相同，都包括2个内含子和3个外显子，而杨树、大豆、拟南芥和蒺藜苜蓿等双子叶植物的SGR基因组结构却含有3个内含子和4个外显子。同源比对结果显示LpSGR基因的核酸序列与BdSGR、OsSGR、ZmSGR、SbSGR和AtSGR的相似度分别为82%、81%、80%、78%和67%。 过量表达LpSGR促进了野生烟草（Nicotiana benthamiana）及拟南芥（Arabidopsis thaliana ）叶绿素降解；LpSGR和AtSGR回补了拟南芥sgr突变体叶片及果夹的滞绿表型；LpSGR定位于叶绿体，并能与其他叶绿素降解蛋白(LpNOL、LpNYC1和 LpPPH)在叶绿体中互作，说明与AtSGR功能相同，LpSGR 基因编码脱镁螯合酶（Magnesium-Dechelatase）在叶绿体中不仅可以催化叶绿素a为脱镁叶绿素a的反应，还可以通过与其他叶绿素降解蛋白的互作来调控叶绿素降解。 LpSGR的转录水平随着衰老的进程而增加，比如衰老后期叶片中的LpSGR基因的表达量极显著高于成熟叶片。脱落酸和乙烯通过其信号传导转录因子（LpABF3、LpABI5、ORE1和EIN3）直接识别并激活LpSGR启动子，进而促进LpSGR 基因的表达。而细胞分裂素则可能通过其信号传递转录因子LpARR1、LpARR10和LpARR12与脱落酸信号途径转录因子LpABF3和LpABI5的互作，间接地抑制LpSGR 基因的表达。褪黑素直接或间接地抑制了LpSGR基因的表达，从而延缓了在黑暗和高温诱导条件下多年生黑麦草的叶片黄化。 通过RNA干扰LpSGR抑制了多年生黑麦草叶绿素的降解通路。在黑暗离体处理8天后，转基因多年生黑麦草叶片中叶绿素含量显著高于野生型。由于LpSGR可以和光捕捉系统蛋白形成聚合体，因此RNA干扰LpSGR可以延缓光捕捉系统蛋白的降解。与野生型多年生黑麦草相比，转基因株系叶片中的粗蛋白含量、体外干物质可消化率和粗脂肪含量显著升高，而转基因株系叶片中的酸性洗涤纤维、中性洗涤纤维、游离镁离子含量和粗灰分含量显著降低。这些结果说明LpSGR不仅参与调控叶绿素降解，还影响了多年生黑麦草饲草品质变化。 RNAi:LpSGR转基因多年生黑麦草与野生型转录数据分析结果表明，RNA干扰LpSGR改变了植物激素信号传导途径，进而延缓了叶片衰老进程；野生型多年生黑麦草衰老叶片中和衰老相关的脱落酸、乙烯和茉莉酸信号传导通路基因表达与成熟叶片相比显著上调；细胞分裂素响应转录因子B-ARRs和A-ARRs转录因子也显著上调；在RNAi:LpSGR转基因株系衰老叶片中脱落酸、乙烯以及茉莉酸等衰老促进激素响应转录因子的基因表达与成熟叶片相比显著下调，这与转基因株系具有缓慢的衰老速度相吻合。 RNA干扰LpSGR改变了多年生黑麦草衰老过程中光合代谢、蛋白合成代谢、脂质代谢、以及饱和、非饱和脂肪酸合成代谢等通路基因的表达。这些代谢通路的改变可能导致了RNAi:LpSGR转基因株系具有高饲草品质等优点。 本研究为滞绿基因LpSGR在多年生黑麦草分子育种上的应用奠定了良好基础。

Leaf yellowing is the common hallmark of leaf senescence. Stress-induced chlorophyll (Chl) and protein degradation promoted premature of leaf senescence. The promoted premature of leaf senescence not only decreased the turf quality, such as shorted the green period of canopy, but also reduced forage quality. Therefore, blocking the Chl degradation by RNA interference Chl degradation genes in perennial ryegrass (Lolium perenne L.), which can be used as turf and forage, technically could delay leaf yellowing and increase its forage quality. Analysis of the regulation mechanism of Chl degradation during leaf senescence is essential for molecular breeding process of perennial ryegrass. In this study, we cloned a SATY-GREEN gene (LpSGR) from perennial ryegrass, which functions as a master regulator in Chl degradation pathway. We also determined its function and mechanism on the hormonal-regulation of Chl degradation. The STAY-GREEN gene, LpSGR, was cloned according to the known SGR genes from rice (Oryza sativaL.) and Arabidopsis (Arabidopsis thaliana) using RACE-PCR method. Its NCBI GeneBank accession number is AQM55942. The coding sequence (CDS) of LpSGR is consisted by 864 bp of nucleotides and coding a protein contains 287 amino acids. LpSGR protein has a typical SGR motif. The genomestructure of LpSGR contains two introns and three exons, which is the same as SGR genes in brachypodium, rice, sorghum, and maize, while deferent from SGR genes in poplar (Populus spp.), soybean (Glycine max L.), Arabidopsis, and medicago (medicago truncatula), which have three introns and four exons. The CDS aliment results indicated that LpSGR shared 82%, 81%, 80%, 78%, and 67% nucleotide sequence similarity to that of BdSGR, OsSGR, ZmSGR, SbSGR, and AtSGR respectively. Overexpression of LpSGR promoted the Chl degradation in wild tobacco (Nicotiana benthamiana) and Arabidopsis. Both LpSGR and AtSGR rescued the stay green phenotypes of leaves and seedpods of Arabidopsis sgr mutant. LpSGR was localized in chloroplast, and it was also physically interactive with other Chl degradation proteins, such as LpNOL, LpNYC1, and LpPPH, in chloroplast. These results indicated that, the same as AtSGR, LpSGR coding a Magnesium-Dechelatase, which not only catalyzes the degradation of Chl a to pheophytin a, but also regulates Chl breakdown pathway through physicially interactive with orther Chl-catabolism enzymes (CCEs). The transcription of LpSGR was positively regulated by the senescence signaling, which was manifested by its relative expression level was almost 1000 folder higher in senescent leaf than that in expanding leaf. Transcription factors in ABA and ethylene signaling pathway, such as LpABF3, LpABI5, ORE1, and EIN3, can enhanced the activity of LpSGR’s promoter by directly binding to its promoter, and further increased the transcription of LpSGR. Transcription factors in CK signaling pathway, such as LpARR1, LpARR10, and LpARR12, can’t directly recognize the promoter of LpSGR, but they can physically interactive with ABA signaling pathway transcription factors, such as LpABF3 and LpABI5. Therefore, CK may inhibited the expression of LpSGR by indirectly regulated its promoter’s activity through the interaction with LpABF3 and LpABI5. Melatonin treatment delayed dark- and heat-induced leaf yellowing by directly or in directly regulated the expression of LpSGR. RNA interference of LpSGR blocked the Chl degradation pathway in perennial ryegrass. Under dark treatment, Chl contents in transgenic lines were significantly higher than that in wild type. SGR protein can combined with the light harvesting complex (LHC) proteins; therefore, RNA interference of LpSGR can delayed the degradation of LHC proteins. Compared to wild type, the crud protein content, in vitro dry matter digestibility (IVTDMD), and total fat content were significantly higher in transgenic lines. The Magnesium (Mg) content, acid detergent fiber (ADF), neutral detergent fiber (NDF), and crude ash (ASH) content were significant lower in transgenic lines than that in wild type. These results indicated that LpSGR not only regulates Chl degradation, but also affects the forage quality in perennial ryegrass during leaf senescence. The transcriptomic changes in RNAi:LpSGR transgenic lines and wild type indicatethat RNA interference of LpSGR altered the transcription of genes which involved in plant hormone signaling pathways. The changed plant hormone signaling pathways may delay leaf senescencein transgenic lines. The transcription of ABA, ethylene, and JA signaling genes were significantly higher in senescent leaves than mature leaves in wild type perennial ryegrass. B-ARRs and A-ARRs are two types of transcription factors in CK signaling pathway were also up-regulated during leaf senescence. However, the transcription of ABA, ethylene, and JA signaling genes were significantly down-regulated in transgenic lines during leaf senescence. These results were consisted with the phenotypic and physiological changes of transgenic lines and wild type during leaf senescence. RNA interference of LpSGR also altered the expression of photosynthesis, protein synthesis, lipid metabolism, and saturated or/and unsaturated fatty acid metabolism related genes. These changes may lead to the increase of crud protein content and forage quality in RNAi:LpSGR transgenic lines. This study provided afundamental knowledge on manipulating STAY-GREEN genes for molecular breeding of perennial ryegrass.