【作者】 王敏；

【导师】 刘继红；

【作者基本信息】 华中农业大学 ， 果树学， 2018， 博士

【摘要】 低温严重影响栽培柑橘的产量、品质以及栽培范围,采用基因工程是创制抗寒柑橘种质的有效手段,利用抗寒资源开展低温应答机制解析及发掘和鉴定抗寒关键基因是重要的基础与前提。枳（Poncirus trifoliata（L.）Raf.）是柑橘的近缘属植物,主要用作柑橘砧木。经低温驯化后,枳可以耐-26 ℃的低温,是极好的耐低温种质资源,但其抗寒机理尚不清楚。本研究基于RNA-seq构建枳低温应答表达谱,筛选到一批低温应答相关的基因。在此基础上,对两个抗寒基因PtrERF109与PtrTZF1进行功能鉴定及抗寒机理解析。主要结果如下:1.对枳进行多种胁迫（4 ℃、干旱、盐）处理,混样建库后使用RNA-seq双末端测序,得到6800万条（5.4 Gb）高质量的clean reads。经de novo组装得到77292条Unigenes,平均长度为1112 bp（N50=1778 bp）。分别有58001和36445条Unigene注释到无冗余蛋白质数据库Nr和Swiss Prot。45819条Unigene可分配到61类GO term中,其中26.3%的Unigene分布于“response to stimulus”term,23846条Unigene可聚类于25类COG term中。KEGG分析表明,这些基因可能参与128条代谢通路。随后对经低温处理（4 ℃,0 h、6 h、24 h、72 h）的枳实生苗叶片采用RNA-seq单末端测序,每个样品各测得约720万条clean reads,分别将这些样品的clean reads比对至上述拼接的Unigene上,并对基因的表达量进行计算。以0 h基因表达量为参考,6 h、24 h及72 h的差异表达基因数分别为600个（462个上调,138个下调）、2346个（1631个上调,715个下调）和5177个（2702个上调,2475个下调）。差异表达基因显著性富集的GO term是“response to chitin”,“response to stimulus”,“response to stress”等。而“plant hormone signal transduction”,“plant-pathogen interaction”及“lipid metabolism”则是显著性最富集的几种代谢通路。差异表达基因中包括60个转录因子,其中以AP2/ERF类转录因子最多。分析还发现,许多差异表达基因与激素代谢和信号传递有关。参与多胺合成的精氨酸脱羧酶基因（ADC）受低温上调,与低温胁迫下的腐胺一致。2.PtrERF109是典型的AP2/ERF类转录因子,属于ERF亚家族B-4亚组。该基因不含内含子,编码308个氨基酸。PtrERF109具有典型的AP2结构域,定位在细胞核,具有转录激活活性,且激活区域位于C端。PtrERF109受低温、脱水和乙烯诱导,但不受ABA和盐诱导。超表达PtrERF109明显增强转基因烟草和柠檬的抗寒性,而枳PtrERF109干涉植株对低温更敏感,表明PtrERF109在植物抵御低温胁迫中起正调控作用。对柠檬转基因植株进行RNA-seq发现,大量基因表达被上调,如响应逆境、糖代谢、脂质代谢、能量代谢等相关基因。其中,5个过氧化物酶基因在超表达柠檬中被上调表达,而过氧化物酶基因Prx1（class III peroxidase 1）是上调倍数最多的基因之一。超表达植株Prx1表达量及过氧化物酶活性低温处理前后均高于野生型,低温处理后的超表达植株积累了更少的H₂O₂,而PtrERF109干涉植株则呈现相反的趋势。进一步通过酵母单杂、凝胶迁移、双荧光素酶分析等证明了PtrERF109结合在Prx1启动子的GCC-box上,并激活其表达,从而介导植物体内ROS清除参与低温应答。3.PtrTZF1不含内含子,编码655个氨基酸,含有典型的串联C3H结构域（即TZF,Tandem Zinc Finger）及ANK结构域,归类于C3H型锌指蛋白家族的第Ⅸ亚家族（拟南芥）或第Ⅰ亚家族（水稻）。该基因受低温与干旱诱导,但受盐和ABA影响较小。PtrTZF1定位在细胞核与细胞质,同时具有转录激活活性及自激活活性,自激活区域在C端。超表达PtrTZF1提高转基因烟草和柠檬的抗寒性,而干涉该基因则降低了枳的抗寒性,暗示PtrTZF1正调控植物的抗寒性。酵母双杂交筛选到多个与PtrTZF1互作的逆境蛋白,包括SPI（Serine protease inhibitor）、b HLH（basic Helix-Loop-Helix）、GRP（Glycine rich protein）、GRP10（Glycine rich protein 10）、ZAT10（Cys2/His2-type zinc-finger protein 10）,其互作得到酵母双杂交和BiFC验证。更多还原

【Abstract】 The yield,quality and cultivation region of commercial citrus were seriously affected by low temperature.Genetic improvement based on genetic engineering is efficient for generating cold resistant citrus varieties,but the important prerequirement and basis are to characterize the mechanism of low temperature response and identify key genes involved in low temperature response Trifoliate orange（Poncirus trifoliata（L.）Raf.）is an closely related genus of citrus and widely used as a rootstock for citrus cultivation.After full cold acclimation,it can tolerate temperature lower than-26 ℃.While understandings about its molecular mechanisms were limited.In this study,the expression profiles of trifoliate orange were constructed based on RNA-seq,and a number of cold-responsive genes were identified.Finally,functions and molecular mechanisms concerning for PtrERF109 and PtrTZF1 in cold tolerance were characterized.The main results are as belows:1.In this study,in order to obtain a general transcriptomic information of trifoliate orange,we significantly expand the breadth of tissue sampling（root,stem,leaf）under different stress treatment（4 ℃,salt,dehydration）.About 68 million（5.4 Gb）high-quality reads were obtained using RNA-seq paired-end sequencing technology.The clean reads were de novo assembled into a non-redundant set of 77,292（>100 bp）Unigenes with an average length of 1,112 bp（N50=1,778 bp）.Sequence similarity search for Unigenes using BLASTX against non-redundant protein databases Nr and Swiss Prot,showed58001 and 36445 Unigenes have homologous sequences in the corresponding library.Of these,23,846 had significant sequence similarity to known genes,and they were assigned to 61 gene ontology（GO）categories and 25 clusters of orthologous groups（COG）.In addition,26.3%（12031/45819）of Unigenes were distributed in the GO term of response to stimulus.KEGG analysis showed that these Unigenes might be involved in 128metabolic pathways.Then trifoliate orange seedlings were treated under 4 ℃,and leaf samples at different time points（0 h,6 h,24 h,72 h）were collected for RNA isolation and library construction.About 7.2 million clean reads were obtained for each liabrary and mapped to the assembled transcriptome,resulting in the identification of 5549differentially expressed genes（DEGs）.These comprised of 600（462 up-regulated,138down-regulated）,2346（1631 up-regulated,715 down-regulated）,and 5177（2702up-regulated,2475 down-regulated）genes from the cold-treated samples at 6,24 and 72 h,respectively.Among GO terms,‘response to chitin’,‘response to stimulus’,and‘response to stress’were the most significantly enriched categories of the DEGs,while‘plant hormone signal transduction’,‘plant-pathogen interaction’,and‘lipid metabolism’were the most significantly enriched metabolic pathways.A total of 60 transcription factors were shown to be cold responsive,among which the number of AP2/ERF transcription factors was the largest.In addition,a number of genes involved in the catabolism and signaling of hormones,such as abscisic acid,ethylene and gibberellin,were up-regulated by the cold stress.Meanwhile,levels of putrescine progressively increased under cold,which was consistent with up-regulation of an arginine decarboxylase gene.2.PtrERF109 is a typical AP2/ERF family transcription factor,belonging to the B-4subgroup of ERF subfamily.This gene is intronless and encodes 308 amino acids.PtrERF109 is located in the nucleus,has transcriptional activation activity,and the activation region is located at the C terminus.PtrERF109 was transiently and strongly induced by low temperature and ethylene,and also induced by dehydration,but not by ABA and salt treatment.Freezing tolerance of transgenic tobacco and lemon overexpressing of PtrERF109 was significantly enhanced.However,silencing of PtrERF109 by VIGS displayed hypersensitivity to freezing stress.These results suggested that PtrERF109 play a positive role in cold tolerance.RNA-seq analysis of the transgenic lemon demonstrated that PtrERF109 overexpression lead a comprehensive gene reprograming in the transgenic plants,in which,genes related to stress response,sugar metabolism,lipid metabolism,and energy metabolism were significantly enriched.Among them,the Prx1（class III peroxidase 1）encoding peroxidas is one of the genes with the highest fold change.The expression level of Prx1 in the overexpressing plants was increased with or without cold treatment,concurrent higher peroxidase activity and less H₂O₂ accumulation in PtrERF109-overexpression plants.By contrast,the VIGS plants showed lower Prx1 level and peroxidase activity,accompanied by increased H₂O₂accumulation relative to wild type.Furthermore,Yeat one-hybrid,EMSA,and dual luciferase assay proved that PtrERF109 could bind to GCC-box in the Prx1 promoter and activate its expression,thereby promoting the scavenging ROS in response to low temperature.3.PtrTZF1 encodes 655 amino acids without an intron,and contains two classical domains at the N terminus,including a tandem C3H（also called as TZF,Tandem CCCH Zinc Finger）and a tandem ANK domain.This protein belongs to the C3H type zinc finger protein,which was further categorized into theⅨsubfamily（Arabidopsis）or theⅠsubfamily（rice）of CCCH zinc finger protein.PtrTZF1 was quickly and strongly upregulated by low temperature,but continuously and slowly induced by dehydration.However,salt and ABA treatment did not cause dramatic alteration of the transcript.PtrTZF1 possessed a strong transcriptional activation and self-activation activity,and the C terminus was required for self-activation.PtrTZF1 was located in both nucleus and cytoplasm in tobacco epidermal cells.Overexpression of PtrTZF1 enhanced the cold tolerance in the transgenic tobacco and lemon,while silencing of this gene in trifoliate orange compromised cold tolerance,indicating that PtrTZF1 played a positive role in cold tolerance.Several stress related proteins,such as SPI（Serine protease inhibitor）,b HLH（basic Helix-Loop-Helix）,GRP（Glycine rich protein）,GRP10（Glycine rich protein 10）,and ZAT10（Cys2/His2-type zinc-finger protein 10）,were confirmed as interacting proteins of PtrTZF1 by yeast two-hybrid screening system and BiFC.更多还原