Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut（Arachis hypogaea L.） Seeds

Phosphoenolpyruvate carboxylase(PEPC;EC 4.1.1.31) catalyses phosphoenolpyruvate(PEP) to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase(PK;EC 2.7.1.40) catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes(AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5) from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties(E11,Hebeigaoyou,Naihan 1,and Huayu 26).It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging(DAP);whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.     

Molecular Identification and Expression Analysis of GhHYDRA 1 Gene,a Homologue of HYDRA1 Gene From Upland Cotton （Gossypium hirsutum L.）

Beside as precursors of BRs biosynthesis,more and more evidences supposed that phytosterols play an important role in plant growth and development.To investigate the effects of phytosterols on the fiber development of upland cotton(Gossypium hirsutum L.)and the molecular base of sterol regulating cotton fiber growth,a homologue of HYDRA1 was cloned from upland cotton(cv.Xuzhou 142)by screening cotton fiber EST database and contigging the candidate ESTs.The GhHYDRA1 encoded a polypeptide of 218 amino acid residues and the deduced amino acid sequences had high homology with the members of HYDRA1 in Populus trichocarpa,Solanum tuberosum,and Arabidopsis thaliana.Moreover,GhHYDRA1 had comparable transmembrane regions to AtHYDRA1 in sequence,length,order,and spacing,except for a C-terminal polylysine cluster.Quantitative real-time RT-PCR analysis revealed that the higher expression levels of GhHYDRA1 gene were detected in 6 to 12 DPA(days post anthesis)fibers,while the lower levels were observed in 0 DPA ovule(with fibers)and 16 to 18 DPA fibers.These results indicated that GhHYDRA1 is the homologue of HYDRA1 gene and plays a crucial role in fiber elongation.Furthermore,auxin and BL up-regulated the expression level of GhHYDRA1 while ABA and KT down-regulated the expression level of GhHYDRA1 in cotton ovule and fiber growth.The result suggested that phytosterols play a role in the interaction of plant hormones.     

Construction of Genetic Linkage Map Based on SSR Markers in Peanut （Arachis hypogaea L.）

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci （QTLs） for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines （RILs） derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut （Arachis hypogaea L.）. A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut （Arachis hypogaea L.）. The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.     

Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton （Gossypium hirsuturm L.）

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene （GhDWF1） involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton （Gossypium hirsutum L.） cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5＇-untranslated region, an ORF of 1 692 bp, and a 120 bp 3＇-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD- dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA （day post anthesis） ovule （with fibers） while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules （with fibers） of wildtype （Xuzhou 142） than in ovules of fuzzless-lintless mutant at the same developmental stages （0 and 4 DPA）. The results suggest that the GhDWF1 gene plays a crucial role in fiber development.     

Gene Cloning and Tissue-Specific Microplitis mediator （Hymenoptera： Expression of G Protein β Subunit in Braconidae）

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut (Arachis hypogaea L.) Seeds

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyses phosphoenolpyruvate (PEP) to yield oxaloacetate, which is involved in protein biosynthesis. Pyruvate kinase (PK; EC 2.7.1.40) catalyzes PEP to yield pyruvate, which is involved in fatty acid synthesis. In this study, five PEPC genes (AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5) from peanut have been cloned. Using a quantitative real-time RT-PCR approach, the expression pattern of each gene was monitored during the seed development of four peanut varieties (E11, Hebeigaoyou, Naihan 1, and Huayu 26). It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging (DAP); whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou. In Naihan 1 and Huayu 26, the expression levels of the five genes remained relatively high in the first stage. The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development. The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter. These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.     

花生长链酰基辅酶A合成酶6基因(LACS6)的克隆、鉴定与组织表达

长链酰基辅酶A合成酶(long chain acyl-CoA synthetase:LACS)是油脂代谢的重要催化酶。为揭示花生脂肪酸代谢机理,采用RT-PCR技术,首次从花生Arachis hypogaea L.克隆到LACS6(GenBank登录号:KU301860),分析该基因的结构组成,预测编码氨基酸与其他植物的同源性,采用Real-Time PCR技术对LACS6的组织表达进行研究。结果显示,花生LACS6基因全长2 116bp,包含2 088bp的ORF,编码695个氨基酸,有23个外显子和22个内含子。氨基酸序列比对显示花生LACS6有真核生物酰基辅酶A合成酶保守结构域,并含有保守的激活位点和绑定位点。同源性分析发现花生LACS6与鹰嘴豆、绿豆、大豆、梅等13种物种的氨基酸一致性在79%~87%,进化树分析显示,花生LACS6与鹰嘴豆等豆科植物亲缘较近。实时荧光PCR分析表明,花生LACS6在花生根、茎、叶、子房柄、仁和花等组织均有表达,且差异明显。子房柄和花的表达量极高,与根、茎、叶和仁等组织有极显著差异,花生LACS6组织的表达量大小排序为花子房柄叶仁茎根。本研究结果为揭示花生脂肪酸代谢和品质改良提供理论依据。     

栽培种花生EST-SSR引物的开发及应用

从高油抗青枯病花生新品系06-4104构建的5个cDNA文库获得了63207条EST,经序列拼接,获得14547条Uni-EST。经MISA检索,在这些Uni-EST中共检测出2643个SSR位点,分布于2250条EST中,发生频率为15.5%,平均每3.1kbEST序列含有一个SSR位点。其中,三核苷酸重复类型出现频率最高,占SSR总数的49.1%,其次是二核苷酸重复类型,占SSR总数的45.8%。在发现的46类重复基序中,AG/TC重复基序的频率最高,AAG/TTC次之。利用Primer3从含有SSR的2250条EST中共设计引物100对,利用这些引物检测花生栽培品种的多态性。结果表明,在所设计的100对引物中有91对在供试的6个花生栽培品种中得到有效扩增,其中13对得到了多态性的扩增产物,每对引物检测出的等位基因数2～3个,平均2.2个。     

基于顺序GISH-FISH花生栽培种的染色体分析

【目的】针对花生染色体较小,染色体细胞学标记少,细胞遗传研究相对滞后,染色体分类识别困难的问题,建立能够准确区分栽培花生（Arachis hypogaea L.,2n=4x=40,AABB）A、B染色体组的新核型,提高染色体识别准确率,以揭示栽培花生和野生供体亲本的染色体对应关系,鉴定栽培种花生染色体结构变异体。【方法】以花生栽培种（Arachis hypogaea L.,2n=4x=40,AABB）的2个可能供体亲本即花生野生种Arachis duranensis（2n=2x=20,BB）和Arachis ipaënsis（2n=2x=20,AA）全基因组DNA及5S rDNA和45S rDNA为探针,利用顺序基因组荧光原位杂交（GISH）和多色荧光原位杂交（McFISH）技术（简称顺序GISH-FISH）结合DAPI染色,在准确区分花生栽培种A、B染色体组的基础上,对花生栽培品种Z5163及其供体亲本染色体进行分析,建立花生栽培种新核型,并利用该核型对其他栽培品种的染色体进行分析,以探讨该核型的应用潜力和栽培花生染色体组成特点。【结果】以A. ipaënsis和A.duranensis全基因组DNA为探针的GISH分析表明,以A. ipaënsis为探针在花生栽培种20条B组染色体上能够产生清晰稳定的杂交信号,在A组染色体上没有信号,而以A.duranensis为探针,只在18条A组染色体能产生信号,但1对A组的小染色体“A染色体”不易被区分,因此,以A. ipaënsis为探针可以准确区分花生栽培种A、B染色体组;综合5S rDNA和45S rDNA Mc-FISH和DAPI染色分析,发现花生栽培种A、B染色体组DAPI带纹、5S rDNA和45S rDNA的分布分别与A.duranensis和A. ipaënsis一致,此结果支持A.duranensis和A.ipaënsis是花生栽培种的供体亲本。DAPI染色结果显示,A. ipaënsis及花生栽培种的B组染色体均有14条染色体显示着丝粒带纹,明显多于前人报道,表明仅利用DAPI染色来区分花生栽培种A、B组染色体的方法具有局限性。综合DAPI染色、rDNA、A.duranensis和A. ipaënsis基因组探针进行顺序GISH-FISH分析,建立了可以准确识别花生栽培种A、B染色体组新核型。然后利用该核型对3个栽培种品种的染色体组成进行了分析,首次发现一个自发的花生染色体代换系MS B1（A1）,揭示了栽培花生染色体B1与A1之间存在部分同源关系。【结论】野生花生A. duranensis和A. ipaënsis分别与栽培花生A和B基因组染色体间具有很好的对应关系;研究建立的基于GISH-FISH和DAPI染色的栽培花生新核型,不但可以准确区分大部分A、B组染色体,而且还能识别栽培花生在多倍体化和人工进化过程中可能存在的自发的染色体变异,揭示A、B组染色体间的部分同源性。     

花生AhbZIP4基因克隆及其在侧枝发育中的表达分析

花生侧枝发育决定株型的建成,但有关花生侧枝发育的基因尚知之甚少。为探究AhbZIP4基因序列特征及在2种株型花生品种的组织器官中的表达模式,本研究从侧枝直立型花生‘冀花5号’（JH5）中克隆获得AhbZIP4基因（Arahy.CS824N.1）序列,并对其进行序列特征分析、亚细胞定位和不同株型品种组织器官表达分析。序列分析表明,该基因编码序列全长1 182 bp,编码393个氨基酸,预测蛋白质分子质量为42.10 kD,蛋白质二、三级结构具典型的bZIP拉链,无跨膜域和信号肽;系统进化分析发现,AhbZIP4与大豆GBF2和GBF2A同源性最高;亚细胞定位显示,AhbZIP4位于细胞核和细胞膜中;表达量分析说明,JH5和匍匐型花生M130除在结荚期时的花中表达差异不显著,在其他生育期的不同组织中均呈现显著或极显著表达差异。其中,2个品种在侧枝发育的15～25 d之间,AhbZIP4表达差异极显著,推测该基因可能间接影响花生株型的分化。研究结果将为进一步揭示花生侧枝发育及株型建成的分子机制提供理论基础。     

中国花生发展及主产区的演变特征分析

20世纪80年代以来,我国花生主产区发生了较大的演变,并表现出一些明显的特点。分析花生主产区演变特征,对于指导生产及政策制定具有重要意义。通过对解放以来中国花生生产情况的分析认为:我国花生种植面积扩大空间变小,提高单产和品质是主产区花生产业发展的根本出路。随着产业的发展, 优质专用花生品种种植规模将进一步扩大,主产区花生加工业将深入发展,进而促进花生产业效益的不断提高。     

北海市春季花生品种的适应性研究

[目的]为了选出适合北海本地推广种植的花生(Arachis hypogaea L.)新品种。[方法]对引进的14个花生品种进行比较试验,分别为桂花236号、桂花106号、桂花99、贺油1049、贺油1127、花育43号、柳花紫3号、金桂花8号、油泰228号、红珍珠、Z62/Q198、华油89、闽惠2号、对照种桂花21。[结果]参试品种中闽惠2号、桂花99的产量分别比对照桂花21号增加6.64%、4.24%,且出仁率、百果重表现突出,在抗病性方面表现较强,各方面综合性状好,为北海本地花生品种示范种植的首选品种,可再进行一次生产试验,综合比较再作推广。而贺油1049、华油89、金桂花8号、花育43号、柳花紫3号产量较低,各方面综合性状较弱,建议直接淘汰。[结论]闽惠2号、桂花99两个品种的产量高,综合性状好,抗病强,适合本地进行示范推广种植。     

Construction of Genetic Linkage Map Based on SSR Markers in Peanut(Arachis hypogaea L.)

Molecular genetic maps of crop species can be used in a variety of ways in breeding and genomic research such as identification and mapping of genes and quantitative trait loci (QTLs) for morphological, physiological and economic traits of crop species. However, a comprehensive genetic linkage map for cultivated peanut has not yet been developed due to the extremely low frequency of DNA polymorphism in cultivated peanut. In this study, 142 recombinant inbred lines (RILs) derived from a cross between Yueyou 13 and Zhenzhuhei were used as mapping population in peanut (Arachis hypogaea L.). A total 652 pairs of genomic-SSR primer and 392 pairs of EST-SSR primer were used to detect the polymorphisms between the two parents. 141 SSR primer pairs, 127 genomic-SSR and 14 EST-SSR ones, which can be used to detect polymorphisms between the two parents, were selected to analyze the RILs population. Thus, a linkage genetic map which consists of 131 SSR loci in 20 linkage groups, with a coverage of 679 cM and an average of 6.12 cM of inter-maker distance was constructed. The putative functions of 12 EST-SSR markers located on the map were analyzed. Eleven showed homology to gene sequences deposited in GenBank. This is the first report of construction of a comprehensive genetic map with SSR markers in peanut (Arachis hypogaea L.). The map presented here will provide a genetic framework for mapping the qualitative and quantitative trait in peanut.     

花生每荚种子数相关性状QTL的定位

【目的】花生是重要的油料作物和经济作物,高产一直是花生育种的主要目标,决定产量的因素是单位面积的种子数和仁重。单位面积种子数是种植密度、每株荚数和每荚种子数的乘积。因此,对花生每荚果种子数相关性状进行QTL分析,有助于发掘该性状相关基因/位点,为花生产量相关性状分子育种提供重要的理论依据。【方法】以四粒红×冀农黑3号构建的RIL群体为研究材料,于2018年（E1）和2020年（E2）在河北省保定市河北农业大学清苑试验站（115°30′E,38°40′N）种植鉴定,收获时调查统计单仁果数、双仁果数以及多仁果数表型值,利用河北农业大学花生创新团队实验室构建的高密度遗传图谱,采用QTL Icimapping V4.2中的完备区间作图法对2个环境下的每荚种子数相关性状进行QTL定位与分析。【结果】单仁果率与双仁果率均呈正态分布,多仁果率呈偏正态分布。3个性状的QTL定位分析结果表明,共检测到11个QTL,可解释4.66%—22.34%的表型变异,加性效应为-9.35—9.42。其中,定位到5个多仁果率QTL,可解释3.19%—22.34%的表型变异,有1个QTL的加性效应为负值（-4.77）,来自冀农黑3号,其余4个QTL的加性效应为正值（3.59—9.42）,均来自母本四粒红;定位到2个单仁果率QTL,可解释4.97%—6.43%的表型变异,加性效应均为负值（-4.45和-4.54）,均来自冀农黑3号;定位到4个双仁果率QTL,可解释3.46%—20.87%的表型变异,加性效应均为负值（-9.35—-3.84）,均来自冀农黑3号。这些QTL中,6个为主效QTL,其中,qRMSPA05被重复检测到,且可遗传表型变异为16.58%—17.34%,加性效应为7.69—8.12。【结论】定位6个主效QTL和1个主效稳定的多仁果率QTL,有助于改良花生产量性状,可以作为遗传改良的重要候选区段,用于分子标记辅助选择与精细定位研究。     

栽培种花生株型相关性状的QTL定位

【目的】栽培种花生是世界范围内重要的油料作物和经济作物,其株型相关性状是典型的数量性状,亦是重要的农艺性状,与产量和机械化收获密切相关。对花生株型相关性状进行遗传分析和QTL定位,筛选与之紧密连锁的分子标记,有助于花生的品种保护和品种鉴别,为花生株型分子育种提供重要的理论依据。【方法】以直立型花生品种冀花5号和匍匐型M130为亲本构建的包含321个家系的RIL群体为研究材料,于2016—2018年分别在海南市、邯郸市、保定市和唐山市等7个环境下种植,各个环境均在收获时调查统计花生侧枝夹角、主茎高、侧枝长、株型指数和扩展半径等5个株型相关性状的表型值。同时,利用SSR、AhTE、SRAP和TRAP等分子标记扫描亲本及群体的基因型用于构建分子遗传连锁图谱。最后结合多年多点的表型数据,采用QTL Icimapping V4.2中的完备区间作图法(inclusive composite interval mapping,ICIM)对7个环境下的株型相关性状进行加性QTL和上位性QTL分析。【结果】构建了一张包含363个多态性位点的分子遗传连锁图谱,所有标记被分配到20条染色体和1个未知连锁群;...