胡萝卜atp8基因的克隆表达及细胞质雄性不育的相关性研究

摘 要：线粒体基因重组而形成的异常开放阅读框的表达导致了植物的细胞质雄性不育现象,在这种现象的研究中具有重要的意义。本研究分离克隆了胡萝卜细胞质雄性不育相关的线粒体基因片段,并利用半定量RT-PCR和Real-time PCR技术研究了该基因在不育系和相应保持系中的表达情况。结果表明,与不育系相比,胡萝卜保持系atp8基因序列在30 bp-170 bp之间核苷酸突变很多,且在154 bp-162 bp发生了缺失。Blast分析发现保持系atp8基因与胡萝卜nad6基因的同源性高达100%。表达分析表明,atp8基因在不育系花蕾的前五个时期表达量都明显低于相应的保持系,而在盛花期的表达量与保持系趋于一致。推测atp8基因的异常表达与胡萝卜细胞质雄性不育有着密切关系。     

小麦K、V型细胞质雄性不育系线粒体基因的比较分析

细胞质雄性不育(cytoplasmic male sterility,MS)是高等植物中普遍存在的一种母性遗传现象,跟线粒体基因密切相关。小麦K型和V型细胞质雄性不育性是由于粘果山羊草和偏凸山羊草或易变山羊草的细胞质导入普通小麦中产生的,被认为是最有利用潜力的小麦不育种质,但是其不育性产生的机理还不清楚。本研究在已知小麦K型细胞质雄性不育系和保持系线粒体基因组序列的基础上,比较分析了K型,V型不育系和保持系中33个线粒体基因和22个开放阅读框(open reading frame,orf;K型不育系特有的,被认为是K型不育系的不育候选基因)。结果表明,线粒体基因中除atp6、nad6和rps3在保持系和不育系之间有较大差异外,其它基因在保持系和不育系中都是高度保守的,其一致性达98%以上,atp6和nad6在两个不育系中也是高度一致的;22个orf中只有orf249在K型和V型不育系之间具有一定差异,其余的orf高度一致,说明小麦K型和V型不育系的线粒体基因是高度保守的,推测二者可能具有相似的不育基因或不育机理。研究结果为小麦K型和V型不育系在杂交育种中的利用奠定了一定的理论基础。     

V型细胞质雄性不育小麦线粒体蛋白质的研究

本实验以V型细胞质雄性不育小麦幼穗为材料,用蔗糖衬垫法和两相法成功地分离纯化了小麦幼穗线粒体,并用SDS-PAGE电泳方法结合高灵敏度的银染技术对其线粒体蛋白质进行了比较分析,发现雄性不育系与相应保持系之间有明显差异,不育系(V1A和V2A)均缺少一个分子量为14kD的蛋白质。线粒体与V型细胞质雄性不育的产生可能存在着某     

制备棉花高分子量线粒体DNA的方法

细胞质雄性不育(cytoplasmic male sterility,CMS)是普遍存在于植物中的生物学现象.细胞质雄性不育受核质两套遗传系统的控制,决定不育的胞质基因在线粒体基因组中.高等植物的线粒体在小孢子的发育过程中起着重要作用,并且与CMS密切相关.因此,通过构建线粒体基因组BAC文库,克隆相关的线粒体基因,有助于了解CMS不育的机理.     

水稻滇Ⅰ型和BT型细胞质雄性不育系的PCR标记鉴别

中国用于杂交粳稻生产的细胞质雄性不育系仅有滇Ⅰ型和BT型,它们的不育系产生的花粉都为染败型,而且具有相同的恢保关系、同为配子体不育。因此,基于花粉败育特点和恢保关系等常规技术无法鉴别这两种不育系。本研究利用线粒体特异引物LD24对27个滇Ⅰ型不育系和9个BT型不育系进行PCR扩增,结果显示滇Ⅰ型不育系无扩增片段,BT型不育系具有1个605 bp的片段。该片段的DNA序列与水稻LD型雄性不育细胞质线粒体基因组的一个604 bp片段相似性达99%,该片段包括一个位于rps1基因下游的一段230 bp序列及rps1与orf187基因间隔区上游的一个374 bp序列。本研究的结果说明利用LD24标记的PCR产物可以有效地鉴别水稻滇Ⅰ型和BT型雄性不育细胞质。     

烟草ATP合酶F_0部分4个亚基基因转录本编辑位点分析

RNA编辑是高等植物线粒体基因转录后表达调控的一种重要方式。为探究ATP合酶F_0部分的4个亚基基因与植物雄性不育性的关系,本研究以3个烟草雄性不育系(MS中烟90、MS云烟85和MS K326)及其同型保持系为供试材料,比较分析atp6、atp9、orf25和orf B线粒体基因转录本的编辑位点。结果表明,orf25和orf B基因转录本在不育系和保持系中发生的编辑位点是一致的。atp6基因在不育系中未发生编辑,在保持系中共有6处发生了RNA编辑。与保持系相比,atp9基因在不育系中除8处共同的C→T编辑外,还缺少2个C→T的特异编辑位点,其中1个导致氨基酸类型的改变。推测不育胞质因缺少特异的线粒体基因转录本编辑而导致烟草的细胞质雄性不育。     

小麦T型细胞质雄性不育保持系线粒体DNA片段转化不育系的初步研究

细胞质雄性不育在植物界普遍存在,其不育机理一直是人们研究的热点。采用花粉管通道法,利用T型细胞质雄性不育保持系75-3369B线粒体的基因文库,选取其中所载外源片段较大的重组子转化T型不育系小麦75-3369A,结果重组子ME252和ME317的转化后代变成可育,表明线粒体的部分片段可以使细胞质雄性不育系的育性发生转变。     

小麦K-型细胞质雄性不育系COI1基因的克隆与表达分析

COI1(coronatine-insensitive 1)基因是茉莉素信号传导中起关键作用的基因,与植物的抗病性、花粉育性密切相关。为探讨COI1与小麦K型细胞质雄性不育性之间的关系,本研究从小麦K型细胞质雄性不育系豫麦3号中同源克隆得到COI1基因的三个拷贝。该基因大小为2.8 kb,开放阅读框为1 761 bp,编码586个氨基酸残基的蛋白,含有COI1蛋白典型的F-box和LRR保守结构域。进化分析结果表明COI1基因的三个拷贝分别与来自小麦祖先种乌拉尔图小麦、拟斯卑尔脱山羊草和粗山羊草、大麦以及短柄草的COI1亲缘关系较近,而与拟南芥、大豆、黄瓜等的COI1亲缘关系较远。利用中国春缺体四体系统进行染色体定位分析,发现它们分别定位于小麦4A、4B、4D染色体上。对COI1基因在小麦品种中国春的根、茎、叶和幼穗中的表达模式分析发现三个拷贝呈组成型表达,但幼穗中Ta COI1A的表达量明显高于Ta COI1B和Ta COI1D。在不育系、保持系和F1代不同发育时期的花药中,COI1基因的三个拷贝在不育系单核期花药的表达量明显高于保持系,在二核期至三核期(不育系花粉败育的关键时期),Ta COI1B和Ta COI1D在不育系和保持系中的表达趋势一致,而Ta COI1A在不育系中的表达呈上调趋势,在保持系和F1代中的表达呈下调趋势,说明Ta COI1A基因在不育系和可育系(保持系和F1代)花药中的表达有明显差异,可能跟不育系的花粉败育有关。     

水稻细胞质雄性不育分子生物学研究进展

对水稻细胞质雄性不育与叶绿体基因组、线粒体基因组、质粒基因组和核基因组的遗传以及育性相关基因的分子标记定位、辅助选择、克隆与表达研究现状进行了综述，并对水稻细胞质雄性不育分子机理和分子生物学研究进行了展望。     

GAPDH基因表达与小麦生理型雄性不育花药败育的关系

为分析三磷酸-甘油醛脱氢酶(GAPDH)基因与小麦(Triticum aestivum)生理型雄性不育花药败育的关系,本研究以新型小麦化学杀雄剂SQ-1作为诱导剂,普通小麦西农1376为材料,构建了西农1376不育和可育等生理系;采用半定量逆转录聚合酶链式反应(RT-PCR)技术分析了不育和可育等生理系不同发育时期花药中GAPDH基因的表达模式.结果表明,该基因在正常小麦花粉发育的单核期、二核期和三核期,表达丰度比较一致,没有明显变化,但在化学杀雄剂SQ-1诱导的生理型雄性不育花药中,不同发育期表达量存在显著差异,单核期表达最弱,二核期最高.与同期正常花药相比,GAPDH基因在生理型雄性不育花药三个发育期均呈下调表达,其中在大量花粉粒败育的单核期,表达量下调最显著,其次为三核期和二核期,证明化学杀雄剂SQ-1对GAPDH基因的表达具有明显抑制作用.因此,化学杀雄剂SQ-1诱导小麦生理型雄性不育形成过程中,可能与GAPDH基因表达减少导致细胞中能量供给不足有一定关系.     

Evaluation of drought and heat tolerance in wheat based on seedling traits and molecular analysis

Polyethylene glycol and cell membrane stability (CMS) assay were used to evaluate drought and heat tolerance, respectively, among 14 wheat lines based on seedling traits and molecular analysis. Significant variation was evidenced for all the investigated seedling traits. Different levels of heritability and genetic advance were found among the tested traits, indicating whether the trait is controlled by additive or non-additive gene action. Drought caused a significant reduction in root and shoot lengths. However, root/shoot ratio under drought stress was increased. Root length showed a highly significant negative correlation with drought susceptibility index (DSI) under drought conditions. Cluster analysis based on seedling traits separated lines mainly by DSI and CMS. Some lines showed drought and heat tolerance by exhibiting a low DSI with high CMS. Sequence-related amplified polymorphism (SRAP) generated a total of 135 bands, with a level of polymorphism ranging from 30 to 86% among the tested lines. SRAP showed its efficiency in discriminating wheat genotypes by gathering all high-DSIlines in one sub-cluster and generating 10 and 3 unique and specific bands for high-DSI-lines and low-DSI-lines, respectively. These bands could be used for further work as SRAP markers associated with drought tolerance in wheat.     

油菜Nsa CMS候选恢复基因的来源及表达

根据同源序列法克隆的Nsa CMS候选恢复基因PPR618的序列,采用PCR方法在Nsa CMS不育系、恢复系、原始体细胞杂交亲本以及其他甘蓝型油菜品种中共克隆出22个同源序列。序列分析表明,野芥亲本野油18、甘蓝型油菜亲本中双4号各含有2个同源序列,而Nsa CMS 4个恢复系则分别含有3、1、1、1个同源序列。恢复系中候选恢复基因的序列与野芥亲本野油18的同源序列的一致性在93%以上,其中恢1、恢3和恢4中至少有一个同源序列与野油18的第一个同源序列完全相同,恢2中的同源序列与野油18的第2个同源序列完全相同;但与甘蓝型油菜的同源序列一致性均低于80%,说明候选恢复基因来源于新疆野芥,而不是甘蓝型油菜。除了原来的PPR618外,获得了3个来源于恢复系的新候选恢复基因。候选恢复基因在序列上与萝卜和矮牵牛的恢复基因一致性较高。半定量RT-PCR分析表明,候选恢复基因在恢复系中多个组织都有表达,但在根、茎中表达量特别少。随着营养器官到生殖器官的发育逐渐升高,候选恢复基因在恢复系中表达量最高的是雄性败育关键时期, 即1.5~2.5 mm的花蕾中,但不育系中的同源基因在茎中的表达量则相对高于其他组织。     

小麦质核互作型雄性不育系线粒体DNA变异性的RAPD分析(英文)

在杂交小麦选育中,为了更好地利用细胞质雄性不育性,研究与不断挖掘新的不育细胞质类型及其对应育性恢复基因以改良现有不育资源至关重要.基于这一目的,本文对具有山羊草属细胞质的四类不育系线粒体DNA(mtDNA)进行了RAPD分析,分别比较了具有同一细胞质背景的山羊草、雄性不育系,以及该类不育系与恢复系组配的可育杂种F1的mtDNA的变异性.结果显示,供试山羊草与其对应细胞质雄性不育系在mtDNA上存在明显多态性,表明不育系在质核互作的影响下很可能已导致mtDNA发生变异:而不育系与对应的可育杂种F1在mtDNA上也存在多态性,同样表明育性恢复核基因对不育系进行育性恢复的过程中亦可能引起mtDNA发生相应变异;mtDNA变异很可能涉及到不育系育性本质的改变.     

甘蓝型油菜胞质雄性不育系22A的遗传研究

甘蓝型双低油菜CMS22A是河南省农科院棉油所利用384A中的雄性不育源、采用单株成对连续回交的方法育成的新型双低油菜细胞质雄性不育系。其突出表现为雄性不育彻底且遗传稳定,配合力高,抗病抗倒,品质优良。对CMS22A育性遗传规律研究表明,该不育系属典型的败药型雄性不育系,其雄性不育的遗传受不育细胞质(S)和一对隐性核不育主效基因(rr)的共同控制,属孢子体不育,基因型为S(rr)。     

腺苷二磷酸葡萄糖焦磷酸化酶活性对小麦K、V、T型不育系育性及籽粒形成的影响

为进一步探寻小麦不育系的不育机制和籽粒不饱满的生理机制,以冀5418核基因为遗传背景,对同核异质K、V、T型不育系叶片、幼穗和籽粒中的腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucose pyrophosphorylase, AGPase)活性和淀粉积累量进行了动态观测,并与各自的保持系进行了比较。在雌雄蕊原基分化期, 不育系幼穗中AGPase活性较保持系高9.33~27.94 μmol g^-1 FW h⁻¹, 差异达极显著水平(F=133.81, P<0.0001); 而在四分体期, 不育系幼穗中该酶活性极显著低于保持系(F=13.97~75.20, P<0.0001),差异为4.27~7.44 μmol g^-1 FW h⁻¹。雌雄蕊原基分化期至四分体时期,不育系叶片中AGPase活性较保持系高7.39~80.77 μmol g^-1 FW h⁻¹,差异极显著(F=135.76~5454.28,P<0.0001)。不育系强、弱势粒中总淀粉、直链淀粉和支链淀粉积累量、AGPase平均活性、淀粉含量及直/支比均极显著低于保持系,且这些指标均表现为强势粒显著高于弱势粒。Logistic方程显示,不育系籽粒淀粉积累量的减少主要由淀粉积累速率降低引起;籽粒AGPase活性与淀粉积累速率显著或极显著正相关(r=0.4460~0.7150, P=0.0004~0.0487);灌浆期, 叶片中AGPase活性与光合速率呈负相关(r=−0.28634, P=0.2823)。因此,雄性不育的可能原因是雌雄蕊原基分化期幼穗和叶片中AGPase活性高,幼穗发育所需能量供应不足;而四分体期幼穗AGPase活性低,影响花粉中淀粉积累。不育系对籽粒AGPase活性具有明显的不良胞质效应,降低ADPG供应水平,影响淀粉的积累,以及旗叶AGPase活性对净光合速率的不良影响,是籽粒不饱满的重要原因之一。     

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.     

Identification of the Rf gene conferring fertility restoration of the CMS Dian-type 1 in rice by using simple sequence repeat markers and advanced inbred lines of restorer and maintainer

Cytoplasmic male sterility of Dian‐type 1 (CMS‐D1) was developed 30 years ago in Yunnan. A major gene conferring fertility restoration for the CMS‐D1 system was detected by microsatellite markers in advanced inbred lines consisting of 196 maintainers and 62 restorers developed in breeding programmes of hybrid rice involving the CMS‐D1 system. The gene was mapped between two simple sequence repeat markers, OSR33 and RM228, on chromosome 10, and was temporarily designated as Rf‐D1(t). The genetic distances of the gene to the two microsatellite markers were 3.4 and 5.0 cM, respectively. This linkage was confirmed by using an F2 population derived from a cross between a CMS‐D1 line and a restorer. This study also demonstrated that using OSR33 was reliable and efficient for identification of restoring lines in hybrid rice breeding with the CMS‐D1 system.     

Modified complementation test of male sterility mutants in pepper (Capsicum annuum L.): preliminary study to convert male sterility system from GMS to CMS

The male sterility system in hybrid seed production can eliminate the cost of emasculation and ensure seed hybridity through avoidance of self pollination. GMS and CMS are two types of male sterility system that currently employed in pepper breeding. Conversion from GMS to CMS will increase the male sterility proportion of female parent from 50 to 100%. In this study, segregation analysis of four male sterile mutants consisting of one CMS mutant (CA1) and three GMS mutants (GA1, GA3 and GA4) showed that each had single recessive gene inheritance. A modified complementation test was performed by replacing male sterile mutants with their maintainer line as male parent. The nuclear restorer gene for CMS was independent of all nuclear restorer genes for GMS and all nuclear restorer genes for GMS were independent each other. Further observation on CMS and GMS male sterility loci revealed that GA1 and GA3 had mutated in both nuclear restorer genes for CMS and GMS, while CA1 and GA4 each carried mutation in single male sterility system of nuclear restorer gene for CMS and GMS, respectively. Conversion from GMS to CMS in the case of lines carried mutations in both sterility systems required only S-type cytoplasm donor, while lines carried mutation in single nuclear restorer gene for GMS required not only S-type cytoplasm but also rf allele donors. The important finding is the broader function of maintainer line in certain male sterility system that can be used as a maintainer or restorer line for other male sterility systems. We also confirmed that line CC1 is the general restorer for both CMS and GMS systems.     

Expression of resistance to Atherigona soccata in F1 hybrids involving shoot fly-resistant and susceptible cytoplasmic male-sterile and restorer lines of sorghum

In recent years, cytoplasmic male‐sterility (CMS) has been recognized as a potential danger to the stability of crop production and resistance to insect pests in sorghum. Therefore, the influence of CMS on the expression of resistance to sorghum shoot fly was studied at the ICRISAT, Patancheru, India using the interlard fishmeal technique. The experimental material consisted of 12 restorer, 12 CMS and the maintainer lines, and their 144 F₁ hybrids. Shoot fly‐resistant CMS lines were preferred for oviposition and had more damage because of deadhearts than the corresponding maintainer lines. The hybrids based on shoot fly‐resistant CMS × resistant restorer lines were significantly less preferred for oviposition than the hybrids based on other cross combinations and exhibited the highest frequency (69.1%) of shoot fly‐resistant hybrids. The hybrids based on glossy and trichomed parents had the highest frequency (>90%) of hybrids with glossy and trichome traits, emphasizing the need to transfer these traits into both parents for better expression in the F₁ hybrids. The expression pattern of trichome density, leaf glossiness and leaf sheath pigmentation in the F₁ hybrids and their parents suggested that the interactions between cytoplasmic and nuclear genes possibly control the expression of traits associated with resistance to sorghum shoot fly in the F₁ hybrids.     

MiR156 and its Target Gene TBCC in Flower Development of Gossypium harknessii Cytoplasmic Male Sterile Line and its Maintainer

This study aimed to analyze the expression level and relationship between miR156 and its target gene TBCC (Tubulin binding Cofactor C) in buds of Gossypium at different developmental stage in a cytoplasmic male sterile(CMS) line and its maintainer line, to clone the full-length cDNA of the TBCC gene, and to determine the possible role of the TBCC gene in pollen development. Real-time RT-PCR showed that the expression levels of miR156 gradually increased with the development of pollen between the CMS line and the maintainer line. The expression of GhTBCC was strikingly complementary with the expression of miR156 in the maintainer line, but showed very low expression levels in the CMS line, which demonstrated that TBCC is related to male sterility. The full-length cDNA sequence of TBCC was cloned from the buds of the maintainer line, and the gene was named as GhTBCC. The CDS of GhTBCC is 1713 bp and encodes a predicted protein of 570 amino acids with a predicted molecular weight of 62.79 kDa. Conserved TBCC and CARP domains were observed in the predicted protein sequence. GhTBCC showed 81 %, 80 %, and 77 % sequence similarity to the TBCC protein sequences of Populous trichocarpa, Ricinus communis, and Arabidopsis thaliana, respectively.