Identification and Characterization of Drought Stress- Responsive Novel microRNAs in Dongxiang Wild Rice

MicroRNAs(miRNAs) are non-coding small RNAs, which play important regulatory roles in response to biotic and abiotic stresses. Dongxiang wild rice(Oryza rufipogon, DXWR) can survive in extreme drought environment, but its molecular mechanism of drought resistance is still largely unknown. To further explore miRNA regulatory mechanisms involved in drought resistance, we identified 138 novel miRNAs in DXWR using small RNA sequencing and bioinformatics approaches, and found that the expression levels of 67 novel miRNAs were significantly affected by drought stress. In total, 200 candidate target genes were predicted and annotated for the drought stress-responsive novel miRNAs. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways suggested that most of the target genes were related to metabolism. Stem-loop quantitative real-time PCR(qRT-PCR) results exhibited high concordance with sequencing data, which confirmed that miRNA expression patterns based on small RNA sequencing in the present study were reliable. Meanwhile, qRT-PCR validated the inverse expression patterns between several miRNAs and their target genes. These results will enhance our understanding of miRNA regulatory mechanisms in response to drought stress in DXWR, and can serve as an important reference for the protection and utilization of this valuable genetic resource.     

Roles of lncRNAs in Rice: Advances and Challenges

Long non-coding RNAs(lncRNAs) larger than 200 nucleotides in length are eukaryotic RNAs, and do not encode proteins. lncRNAs are considered to be important regulators in many biological processes in plants. In this review, the latest advances in the roles of lncRNAs in rice were reviewed, and functions of lncRNAs were further summarized and classified. lncRNAs play crucial roles in tolerance to biotic and abiotic stresses such as disease, drought, heat, cold, heavy metal, and starvation of nitrogen and phosphorus in rice. In addition, lncRNAs also regulate various growth and development processes in rice. Finally, the prospect and challenges of rice lncRNA function research in future were proposed. This review will not only help us quickly grasp the frontiers of lncRNAs functional research progresses, but also propose suggestions and ideas for the directions of further lncRNA research in rice.     

转录组测序筛选野生大豆糖代谢途径干旱相关基因及其表达差异分析

野生大豆较栽培大豆有更广的遗传多样性,在组学水平整体分析其基因表达模式对筛选抗旱关键基因、 解析干旱胁迫响应调控网络和促进分子育种具有重要意义。本研究采用高通量测序技术对PEG6000模拟干旱胁 迫处理第0 h、6 h、12 h、24 h和48 h的30d苗龄野生大豆RNA进行转录组测序,获得了1796条糖代谢途径相关序 列。淀粉和蔗糖代谢途径通路注释的差异基因最多,半乳糖代谢途径次之;在获得的109个差异表达基因中,干旱 胁迫处理第12 h差异表达基因最多;对以上109个差异表达基因构建蛋白质相互作用网络,以节点度>10为标准筛 选中心节点,共获得8个关键基因。为进一步研究野生大豆干旱胁迫下的糖代谢相关基因奠定了基础。     

Defensive Role of Plant Hormones in Advancing Abiotic Stress-Resistant Rice Plants

Consistent climatic perturbations have increased global environmental concerns, especially the impacts of abiotic stresses on crop productivity. Rice is a staple food crop for the majority of the world’s population. Abiotic stresses, including salt, drought, heat, cold and heavy metals, are potential inhibitors of rice growth and yield. Abiotic stresses elicit various acclimation responses that facilitate in stress mitigation. Plant hormones play an important role in mediating the growth and development of rice plants under optimal and stressful environments by activating a multitude of signalling cascades to elicit the rice plant’s adaptive responses. The current review describes the role of plant hormone-mediated abiotic stress tolerance in rice, potential crosstalk between plant hormones involved in rice abiotic stress tolerance and significant advancements in biotechnological initiatives including genetic engineering approach to provide a step forward in making rice resistance to abiotic stress.     

水稻幼穗响应稻曲病菌毒素胁迫早期的转录组分析

【目的】由稻曲病菌引起的稻曲病不仅造成水稻减产,而且还会产生对动物和植物有毒的真菌毒素。探明水稻幼穗对稻曲病菌毒素胁迫响应的分子机制,可为发掘水稻抗稻曲病基因以及抗病分子育种开辟新的思路。【方法】用稻曲病菌毒素处理水稻幼穗,采用转录组测序技术对水稻幼穗进行转录组测序,以水稻9311基因组作为参考基因组进行对比,利用TPM法计算基因表达量,设定参数（差异倍数的绝对值不小于2,且q值不大于0.05）筛选差异表达基因。结合基因差异表达分析、富集功能分析,鉴定水稻响应胁迫的关键基因,并利用实时荧光定量PCR技术对差异表达基因进行验证。【结果】在稻曲病菌毒素胁迫12 h后,水稻幼穗出现2526个差异表达基因（DEG）;通过GO富集、KEGG代谢途经和KOG功能分析,将差异基因划分为GO功能下的64个条目、32个代谢途径和KOG功能下23个类别,包括淀粉和蔗糖代谢、苯丙类生物合成、碳代谢、糖酵解/糖异生、氨基糖和核苷酸糖代谢等生物学过程。DEG中有66个植物转录因子,分属7种植物转录因子家族,包括WRKY和Myb两大转录因子。分析二萜类生物合成与淀粉和蔗糖代谢途径相关基因发现,OsCPS2、OsKSL4和细胞色素P450等基因表达量上调,而淀粉酶、β-呋喃果糖苷酶和UDP-焦磷酸化酶等基因表达量下调,推测这些基因在水稻响应稻曲病菌毒素胁迫时发挥重要的作用。【结论】稻曲病菌毒素作为非生物胁迫因素对水稻幼穗具有毒性;通过干扰淀粉和蔗糖代谢等途径而影响种子营养物质的合成,降低水稻抵抗病原菌侵染水稻的能力。     

Hidden diversity for abiotic and biotic stress tolerances in the primary gene pool of rice revealed by a large backcross breeding program

Low and unstable rice productivity in many areas of Asia is associated with many abiotic and biotic stresses such as drought, salinity, anaerobic conditions during germination, submergence, phosphorus and zinc deficiency, etc. To develop rice varieties with tolerance to these stresses, we undertook a large backcross (BC) breeding effort for the last 6 years, using three recurrent elite rice lines and 203 diverse donors, which represent a significant portion of the genetic diversity in the primary gene pool of rice. Significant progress has been made in the BC breeding program, which resulted in development of large numbers of introgression lines with improved tolerance to these stresses. Promising lines have been developed with excellent tolerances (extreme phenotypes) to salinity, submergence and zinc deficiency; resistance to brown plant hopper, ability to germinate under the anaerobic condition and low temperature. Our results indicated that there exist tremendous amounts of ‘hidden’ diversity for abiotic and biotic stress tolerances in the primary gene pool of rice. Furthermore, we demonstrated that despite the complex genetics and diverse physiological mechanisms underlying the abiotic stress tolerances, introgression of genes from a diverse source of donors into elite genetic backgrounds through BC breeding and efficient selection (careful screening under severe stress) is a powerful way to exploit this hidden diversity for improving abiotic stress tolerances of rice. We have developed three large sets of introgression lines, which not only provide an unique platform of breeding materials for developing new rice cultivars with superior yield and stability by trait/gene pyramiding, but also represent unique genetic stocks for a large-scale discovery of genes/alleles underlying the abiotic and biotic stress tolerances of rice using genomic tools.     

A β-ketoacyl-CoA Synthase OsCUT1 Confers Increased Drought Tolerance in Rice

Drought stress is one of the major environmental factors affecting crop growth and productivity. Cuticular wax plays essential roles in protecting plants from environmental stress via forming a hydrophobic barrier on leaf epidermis. In this study, we analyzed nine members (OsCUT1?OsCUT9) of β-ketoacyl-CoA synthase, the rate-limiting key enzyme for cuticular wax synthesis in rice by homology search and domain prediction. The expression levels of OsCUT genes under different abiotic stresses were investigated and OsCUT1 down-regulated by abiotic stress was selected for further function validation. Compared to the wild type, overexpression of OsCUT1 (OX-OsCUT1) exhibited significantly increased drought resistance. Epicuticular wax was increased on the leaf surface of OX-OsCUT1 and the chlorophyll leaching experiment showed that the cuticular permeability was decreased in the OX-OsCUT1 plants. Moreover, overexpression of OsCUT1 didn’t result in the significant changes of major agronomic traits. In total, these results suggested that OsCUT1 is a promising gene for engineering rice plants with enhanced drought tolerance.     

木薯中lncRNA-CRR5在低温胁迫下的功能分析

低温胁迫严重影响木薯的生长、产量以及块根品质。长链非编码RNAs（lncRNAs）作为关键的调节因子,在植物响应非生物胁迫过程中发挥重要的功能。然而,lncRNAs在木薯中的功能和调控机制尚无报道。因此,为了研究木薯lncRNAs在低温胁迫应答中的功能,本研究以木薯主栽品种‘60444’为材料,利用低温处理前后的叶片转录组数据,鉴定到一个受低温胁迫调控的lncRNA（cold-responsive lncRNA5,CRR5）。通过对其序列比对进行同源性分析, RNA二级结构预测和共表达分析鉴定来分析lncRNA-CRR5响应低温胁迫的分子功能。并根据基因的共表达分析推测CRR5的顺式作用和反式作用靶基因。这些结果为更进一步研究CRR5的功能提供了理论依据。     

燕麦植物激素信号转导通路中响应干旱胁迫的关键基因

植物激素信号转导通路是响应胁迫的重要通路，该通路中基因的表达调控作物的抗旱性。为挖掘燕麦植物激素信号转导通路中响应干旱胁迫的关键基因，本研究对燕麦幼苗进行不同干旱胁迫处理，取叶片进行转录组测序，分析不同处理下基因的表达。结果表明，与正常水分条件相比，轻度干旱胁迫（PEG 10%）诱导624个基因表达发生显著变化（DEGs），重度干旱胁迫（PEG 20%）诱导13 063个。GO富集分析显示，重度干旱胁迫下，DEGs主要富集在对胁迫反应的调节；KEGG分析显示，轻度干旱胁迫下，植物激素信号转导通路中1个ARF和2个CRE1基因表达显著下调，表达量较高，可能为燕麦在该通路中响应轻度干旱胁迫的基因；而重度干旱胁迫下，植物激素信号转导通路富集169个DEGs，其中生长素和脱落酸信号转导过程DEGs较多，分别占植物激素信号转导通路总DEGs的20.7%和15.9%；在8条激素信号转导过程中筛选出12个表达量较高的基因，可能为燕麦在该通路中响应重度干旱胁迫的关键基因。本研究将为今后燕麦抗旱基因的克隆和验证提供依据。     

NAC转录因子在水稻抗逆基因工程中的应用进展

水稻经常遭遇干旱、高盐、低温、病原菌等逆境胁迫,影响其生长发育甚至产量。NAC转录因子是植物特有的、最大的转录因子家族之一,在水稻生长发育及抵御多种非生物及生物胁迫反应中具有重要的调控作用。本文阐述了水稻NAC转录因子的结构、分类及染色体定位,并阐述了NAC转录因子在水稻抗旱、耐盐、耐冷及抗病等抗逆基因工程中应用的研究进展,为NAC转录因子的利用及水稻抗逆遗传改良提供参考。     

Screening for Osmotic Stress Responses in Rice Varieties under Drought Condition

Drought is the major abiotic stress factor that limits rice production worldwide.To evaluate the osmotic stress responses in rice varieties under drought condition,a total of 42 high-yielding rice varieties were collected from various research stations of Kerala Agricultural University in India.The experimental setup comprises of initial hydroponic treatments at different osmotic potentials,artificially induced by desired strengths of polyethylene glycol(PEG6000),and followed by the pot planted experiments in the rain-out-zone.The activities of antioxidant enzymes,relative water content,cell membrane stability,photosynthetic pigments,proline content,along with plant growth parameters of the varieties under drought condition were evaluated.Moreover,the standard scores of these rice varieties were assessed under stress and recovery conditions based on the scoring scale of the Standard Evaluation System for rice.Among the 42 rice varieties,we identified 2 rice varieties,Swarnaprabha and Kattamodan,with less leaf rolling,better drought recovery ability as well as relative water content,increased membrane stability index,osmolyte accumulation,and antioxidant enzyme activities pointed towards their degree of tolerance to drought stress.The positive adaptive responses of these rice varieties towards drought stress can be used in the genetic improvement of rice drought resistance breeding program.     

Identification of deletion mutants with improved performance under water-limited environments in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a semi-aquatic member of the grass family that is poorly adapted to dry environments and has greater sensitivity to water-deficits than other important cereals in this family. To increase productivity in aerobic or water-limited environments rice must overcome its adaptations to flooded environments. Deletion mutants offer an alternative genetic resource for improving drought tolerance. Almost 3500 IR64 deletion mutants were screened under vegetative and reproductive stage drought stress in the field and evaluated for leaf drying and/or grain yield. Seven novel conditional mutants of rice which showed gain of function through continued growth as drought stress developed compared to the wild type were identified. Mutant recovery rate was 0.1%. Further evaluation of putative drought mutants revealed that their average shoot biomass at maturity and grain yield per plant under stress exceeded those of the wild type by two-fold. Studies under controlled conditions confirmed mutants to have continued growth of both roots and shoots as drought developed compared to the wild type, and a tendency for greater water extraction. We propose that deletions in these mutants have affected a regulator of the highly conservative growth response common to irrigated lowland rice cultivars. Our results suggest that screening deletion mutants for performance under managed drought stress in the field could be a highly effective way to identify valuable genetic resources for improved drought response and aerobic adaptation in rice.     

Expression pattern of GmLAX genes under different stresses in soybean drought sensitive cultivar and tolerant cultivar

Auxin has been reported to regulate plant growth and development, as well as to mediate plant adaption to abiotic stresses, including drought. AUX/LAX family displays auxin uptake functions and comprises four highly conserved genes AUX1 and LIKE AUX1 (LAX1, LAX2, and LAX3) in Arabidopsis. There are fifteen GmLAX family genes in the soybean genomes and several members were regulated by dehydration stress. In this study, the sequence differences and expression pattern of GmLAXs-I were analyzed under stress treatment between the soybean drought-tolerant Jindou 21 and drought-sensitive varieties Zhongdou 33. Five homologous genes of AUX1 were all responsive to PEG, salt, ABA and IAA stimuli. There were two SNPs in the promoter region of GmLAX4 gene, and this gene was differentially expressed in two cultivars. Moreover, our results showed YFP-GmLAXs are predominantly localized in plasma membrane. Taken together, our results suggest that GmLAXs are involved in abiotic response, which can provide theoretical and technical support for the genetic improvement of soybean drought tolerance.     

干旱胁迫下杂草稻和栽培稻根系基因表达差异研究

利用Affymetrix水稻表达谱芯片(GeneChip Rice Genome Array)研究抗旱杂草稻HEB07-2与巴西陆稻(IAPAR9)在PEG模拟干旱胁迫下根系基因表达变化。结果表明,杂草稻HEB07-2转录组对干旱信号的响应程度与方向均与巴西陆稻存在很大差异,HEB07-2以正向调控为主,而巴西陆稻以负调控为主。干旱胁迫下杂草稻HEB07-2与巴西陆稻分别有6878个和2923个基因表达,其中HEB07-2受干旱胁迫诱导上调的基因有4693个,下调的基因有2185个;而巴西陆稻则分别有983个和1940个。在差异基因表达的倍数上也是HEB07-2高于巴西陆稻。进一步的GO分析表明,在干旱胁迫下,HEB07-2和IAPAR9根系基因响应途径的差异表现为HEB07-2在钾离子转运(GO:0006813)、次生物质代谢(GO:0019748)、细胞生长(GO:0016049)、葡萄糖代谢(GO:0006006)、跨膜离子转运器活性(GO:0015075)、亚铁血红素结合体(GO:0020037)、氧化还原酶活性(GO:0016491)等方面基因显著上调,这与生理数据和表型数据一致。     

Evaluation of Agronomic Traits in Chromosome Segment Substitution Lines of KDML105 Containing Drought Tolerance QTL under Drought Stress

Drought is a major abiotic constraint to rice production in rainfed lowland and insufficiently irrigated areas.The improvement of drought tolerant varieties is one of the strategies to reduce the negative effects of drought.Quantitative trait loci(QTLs) for primary and secondary traits related to drought tolerance(DT) on chromosomes 1,3,4,8 and 9 that determined from double haploid lines derived from a cross between CT9993 and IR62266 were introgressed and dissected into small pieces in the genetic background of Khao Dawk Mali 105(KDML105) to develop chromosome segment substitution line(CSSL) population.The CSSLs were evaluated at the reproductive stage for their agronomic performance and yield components under drought stress,and results were compared with irrigated condition.The flowering of CSSL lines was 6 to 7 d earlier than KDML105.The mean values of grain yields in the CSSLs were higher than KDML105 under drought and irrigated conditions.At irrigated condition,the grain yields of introgression lines carrying DT-QTLs from chromosomes 4 and 8 were higher than that of KDML105,whereas other traits showed little difference with KDML105.Analysis indicated that grain yield has positive correlation with plant height,tiller and panicle number per plant,and total grain weight per plant under drought stress while negatively correlated with days to flowering.As mentioned above,CSSLs showing good adaptation under drought stress can be used as genetic materials to improve drought tolerance in Thai rainfed lowland rice breeding program,and as materials to dissect genes underlying drought tolerance.     

大豆GmFDL06基因抗干旱及耐盐性研究

bZIP转录因子在植物防卫反应和逆境胁迫应答过程中起着十分重要的作用,大豆GmFDL06是bZIP转录因子家族A组成员,其表达水平受PEG和高盐胁迫影响。本研究分析了GmFDL06转基因大豆苗期的抗干旱和耐盐性。PEG和盐处理后GmFDL06转基因植株的相对植株高度(RPH)和相对植株干重(RSDW)显著高于非转基因大豆东农50。在盐胁迫下,GmFDL06转基因植株比东农50伤害程度低,并且GmFDL06过表达减少了Na~+离子积累,降低了盐胁迫带来的伤害,且过表达GmFDL06促进了下游逆境胁迫基因的表达。这些研究结果表明GmFDL06参与大豆非生物胁迫反应,并有潜力改善大豆的干旱和盐胁迫耐受性,为大豆抗逆基因的研究及抗逆大豆材料的筛选提供了依据。     

小麦SPL家族基因在非生物胁迫下的表达分析

SQUAMOSA promoter binding protein like(SPL)是一类在植物中广泛存在的转录因子家族,在调控植物生长发育、响应逆境胁迫等方面发挥着重要作用。为解析小麦中SPL家族基因响应非生物胁迫的机理,本研究采用生物信息学的方法在全基因组范围内对小麦SPL基因家族成员进行鉴定,并对鉴定到的SPL基因进行表达模式分析。结果表明,在全基因组范围内共鉴定到56个小麦SPL基因,其中27个是miR156的靶基因;系统进化分析发现,56个小麦SPL基因聚类为7个亚家族。基于转录组数据对表达模式进行分析,发现36个小麦SPL基因与非生物胁迫响应相关,响应缺氮、缺磷、高盐、低温、干旱、高温胁迫以及热旱共胁迫的基因分别有12、16、22、6、13、14和21个,其中TraesCS3D02G425800同时响应7种非生物胁迫。qRT PCR验证结果与转录组数据基本一致。