木薯sRNA 测序分析及其开花相关 microRNA的挖掘

为研究木薯花序与叶片中 sRNA 表达特性,探索 microRNA 对其开花过程的调控。本研究以木薯品种“华南八号”花序和幼叶为材料,利用 RNA-seq 技术进行 sRNA 测序分析;并筛选开花相关 microRNAs 进行表达特性分析。从花序和幼叶测序结果中分别获得 12 092 109 条和 11 499 655 条 sRNA 序列。花序中筛选出 139 条已知 microRNAs 和253 条新预测 microRNA,分别预测得到 992 和 3 736 条靶基因;幼叶中筛选出 134 条已知 microRNA 和 191 条新预测microRNAs,分别预测得到 979 和 2 603 条靶基因。最终筛选出 8 种与开花调控相关和 3 种花色调控相关的 microRNAs。表达分析显示,miRNAs 在两样品间呈现出较大的整体性差异,并且在功能与代谢通路上也不尽相同;开花相关microRNAs 中表达差异较大的为 miR156、miR172 和 miR169,其中 miR156 表达量在花序中高于幼叶,而 miR172 表达量在花序中低于幼叶,推测其功能为抑制后续的开花过程,避免过度开花。本研究分析了木薯花序与幼叶中 sRNA的整体特性,通过表达差异分析初步明确了 microRNAs 对木薯开花的调控,为进一步深入探索奠定了基础。     

木薯耐寒相关microRNA的差异表达分析

采用荧光实时定量PCR(quantitative Real Time-PCR,qRT-PCR)技术对低温胁迫处理及对照的两个木薯品种C4和KU50材料进行差异表达分析。结果表明,该6个miRNA在胁迫材料与对照中的表达不同,不同品种及器官中也存在差异表达,且6个miRNA属于受低温诱导与上游调节基因相关的miRNA,这将成为今后研究的主要对象。     

苋菜试管开花过程中相关microRNA表达的qPCR分析

以苋菜无菌试管苗及其试管开花过程中各阶段培养物为材料,利用qPCR技术进行苋菜试管开花过程相关microRNA的表达分析。研究结果表明:由NormFinder软件计算出最适合的内参基因为ama-miR159a;在苋菜试管开花过程中,ama-miR156c、ama-miR156d、ama-miR156g相对表达量在幼苗期向壮苗期转变过程中呈下降趋势,并在整个发育过程中一直保持较低水平;ama-miR156h、ama-miR157b、ama-miR157c、ama-miR157d、ama-miR3和ama-miR10相对表达量在幼苗期向花芽期转变过程中呈下降趋势,并在花芽期达到最低;ama-miR172d的表达模式与miR156/miR157表达模式相反;苋菜试管苗幼苗期到花芽期形成时ama-miR319b相对表达量呈上升趋势,ama-miR164b在花芽期时相对表达量比较高,花芽期向开花期转变时,其相对表达量降低;ama-miR166a/miR166g表达在苋菜开花过程中始终呈上调表达;ama-miR167b/miR167d和ama-miR4相对表达量先略有升高然后降低,在花芽期降到最低,随后又升高。由此可见,microRNA在调控苋菜试管苗由幼苗期向壮苗期转变、营养生长向生殖生长转变以及花器官的形成等过程中起重要的调控作用。     

木薯抗旱相关miRNA表达差异分析

对2个木薯(Manihot esculenta Crantz)品种SC124和KU50进行驯化和非驯化干旱处理,利用实时荧光定量PCR技术(qPCR)取样2个部位的3个时间点,对5个miRNA进行表达差异研究。结果显示,5个miRNA具有不同的表达形式,miRNA 171 ab和398 b经过驯化后诱导(DH)条件下表达,miRNA 166 a在直接干旱处理(DNH)条件下的功能叶中有表达,而驯化后诱导条件下只在根中有表达;miRNA 390 b和399 e在2种干旱处理方式中都有表达,经过驯化后表达量也明显增加。通过对不同处理、部位和时间点上的表达情况分析,表明miRNA具有明显的品种特异性、组织特异性和时序性。     

木薯蔗糖转运蛋白基因的表达分析

以木薯SC124为材料,分别在植株发育8个时间点取其块根和功能叶,利用实时定量PCR技术（qPCR）对木薯SUT1、SUT2、SUT4三类蔗糖转运蛋白基因表达差异进行研究。结果表明,在木薯块根形成期（90 d）3类SUT表达的日变化中,功能叶和块根中均以中午12 h最高,其余时间较低,叶片中SUT1和SUT2表达水平较高而块根中差异不显著;在整个发育过程中,叶片表达水平高于块根,且二者均为前期表达水平高,后期下降,3个SUT之间的表达量也有着明显的差异,叶片中SUT1的表达量最高,其次是SUT2,SUT4最低,在块根中除60 d时SUT1的表达量较高外,其它SUT表达量均较低,SUT4在叶片和块根中的表达量差异不大。     

木薯褐斑病病原鉴定及其生物学特性研究

褐斑病是中国木薯生产中最为严重的叶部真菌性病害.采用多种分离方法,经致病性评价后获得该病的病原菌,形态观察和ITS序列分析结果表明其为亨宁氏钉孢(Passalora henningsii).比较光照、菌丝打断、紫外线和菌丝打断-紫外等方法的诱导产孢效果,结果表明:只有菌丝打断-紫外诱导法能够产生大量分生孢子,CME(麦芽木薯叶)培养基、28℃、pH6.0～7.0、紫外线连续诱导、25 mL/皿、14～21 d为产孢最适条件.对菌株CPHHN02的生物学特性研究表明,CA培养基、26～28℃、pH6.0、连续黑暗、碳氮源分别为D-葡萄糖、D-甘露醇、D-乳糖和胰蛋白胨为菌株最适培养条件.大型分生孢子的最适萌发温度为26℃,致死温度为60℃10 min.     

木薯MeHDS2基因的克隆与分析

采用RT-PCR技术,从木薯SC124 cDNA中克隆得到一个具有完整阅读框的基因,命名为MeHDS2,该基因全长2 529 bp,编码842个氨基酸。蛋白质保守结构域分析结果表明,MeHDS2蛋白含有保守的1个HD结构域、1个b-zip结构域、1个START结构域和1个MEKHLA结构域;蛋白质空间结构预测显示其具有3个典型的α螺旋结构;亚细胞定位结果表明,MeHDS2蛋白在植物细胞核中特异表达,因此,推测其为HD-ZIP转录因子家族中的一员。基因表达分析结果表明,MeHDS2基因在木薯根、茎、叶中都有表达,但表达丰度不同。木薯干旱处理14 d后,自形态学顶端往下数第三、四片叶片的MeHDS2表达量显著高于根、茎及其他位置叶片组织。本研究为木薯抗旱分子育种提供了理论基础。     

木薯全粉的功能特性

开发推广木薯源大宗食物,充分发挥木薯的资源优势,拓展人们日常消费的食物资源,积极应对日益严重的粮食危机.通过对木薯全粉与鲜木薯、木薯淀粉中的营养成分的测定对比,及对木薯全粉的黏度特性、透光率、膨胀度等进行分析,并对木薯全粉的营养成分保留及功能性质进行评价,探讨木薯全粉在食品工业中的潜在应用范围.结果表明,木薯全粉相对于鲜木薯、木薯淀粉营养成分保持良好,食用安全,同时具有较好的加工性能,在食品工业中应用前景广阔.     

木薯新叶斑病病原鉴定及其生物学特性

2008和2009年在对木薯主产区病害的普查中,发现了一种新的叶斑病,命名为离孺孢叶斑病。形态观察结果表明,该病原菌分生孢子梗丛生,褐色,基细胞膨大呈半球形,成熟的分生孢子长椭圆形,两端钝圆,具5～8个隔膜,表明其为狗尾草离孺孢。ITS序列分析结果表明其和一株狗尾草离孺孢(EF452444)的序列同源性为99%。生物学特性研究结果表明,PDA培养基、28℃、pH7和光暗交替是菌落生长最适条件,D-葡萄糖和硝酸钠是其最适碳氮源,孢子萌发最适温度和致死温度分别为28℃和55℃5min。10种杀菌剂的室内筛选结果表明,咪鲜胺(25%咪鲜胺EC)的EC50值最小,抑菌效果最好。     

木薯二倍体及其同源四倍体开花结实特性的比较

以木薯品种‘新选048’和‘华南205’的二倍体及其同源四倍体为材料,采用定株观察的方法,比较二倍体及其同源四倍体开花结实特性的差异,探究染色体加倍后对木薯开花结实特性的影响,为开展木薯多倍体材料创制及鉴定提供科学参考。结果表明：2个木薯品种四倍体的开花结实物候期都要迟于二倍体,且四倍体的雌花、雄花、花药、果实、种子的形态大小均大于二倍体;2个木薯品种不同倍性间花药分布情况存在差异,‘新选048’的四倍体会出现两性花且其雌花花被颜色与二倍体存在明显差异;2个木薯品种不同倍性间开花及坐果数量也存在不同程度的差异,其中‘新选048’四倍体的单序总花量和雌、雄花总量显著大于二倍体的,但2个品种在不同倍性间开花及坐果数量的差异中呈现不同的规律。因此,木薯染色体加倍,可延迟开花结实的时间,增加生殖器官的大小,改变花形态特征和雌雄花的数量及花总量,但这种改变具有品种依赖性。     

5个木薯品种染色体核型与聚类分析

本研究对5个木薯品种进行核型分析以揭示其核型特征,通过聚类分析来自4个产地的5个品种间的相似性,以探讨其亲缘关系。实验采用压片法,以木薯嫩叶为材料,运用DPS软件对核型资料按照最长距离法进行聚类分析。结果表明,木薯5个品种的染色体数目均为2n=36,其中,SC12的核型公式为2n=36=34m+2sm,SM2300-1的核型公式为2n=36=36m(4SAT)、桂热5号的核型公式为2n=36=36m,云南8号与ZM8752的核型公式均为2n=36=34m(4SAT)+2sm。核型不对称系数范围在56.58～58.85之间,核型类型依次为1A、1B、1B、2A、1A,对称程度较高。聚类结果显示,在遗传距离为0.4时,5个品种分为3类。第Ⅰ类为云南8号,第Ⅱ类包括ZM8752与SM2300-1,第Ⅲ类包括SC12和桂热5号,5个品种存在一定的核型差异,说明具有丰富的遗传多样性。     

木薯DBE和SBE基因的序列分析及其茉莉酸甲酯诱导表达特性

为研究木薯中调控支链淀粉合成的DBE和SBE基因如何被茉莉酸信号调控,首先对木薯基因组数据库中的MeDBE和MeSBE2.1基因进行序列分析,二者都具有α淀粉酶催化结构域且启动子区都具有茉莉酸响应元件等多种响应元件。以木薯品种‘华南八号’悬浮培养细胞为材料,利用qRT-PCR检测木薯MeDBE和MeSBE2.1基因在茉莉酸甲酯处理后的表达特性。结果显示：MeDBE基因的表达在最初短暂上调后持续下调,而MeSBE2.1则持续上调后又恢复最初水平,说明MeDBE和MeSBE2.1基因可被茉莉酸信号调控,进一步推测表明,其受到不同激素信号整合后的综合调控,以影响支链淀粉的生物合成。     

低温驯化对木薯耐寒性形态、生理特性的影响

用相对耐寒的2个木薯品种华南124和Arg7为材料,设置14℃弱低温驯化后4℃伤害处理、非驯化直接4℃处理和25℃对照,观察和测定了木薯在低温驯化处理下的形态与生理变化。结果表明,该2个品种经低温驯化处理后比非驯化处理,植株形态损伤减轻,恢复正常温度后形态恢复力增强;叶片中的叶绿素含量下降延缓、脯氨酸含量增幅高而丙二醛含量指示的细胞膜破损变小;而在温度恢复过程中叶绿素含量、叶片脯氨酸含量增加均较快。形态与生理指数一致表明,弱低温驯化能够有效提高木薯对低温的适应能力及恢复能力。     

我国木薯加工业的发展现状与展望

木薯作为一种经济作物,具有良好的综合利用价值,其加工利用途径非常广泛。根据我国近几年木薯加工的研究成果,对我国木薯加工业的发展现状进行了概述,指出了我国木薯加工在原料、加工技术、产业等方面存在的一些问题,并就此提出几点建议。     

木薯MeNRT2.1基因的克隆及表达分析

木薯具有产量高、抗贫瘠等特点,为了了解其耐贫瘠的作用机理,提高木薯在贫瘠土壤中对氮素的利用率,以培养20 d的"华南5号"木薯组培苗为实验材料,采用同源克隆和RT-PCR技术,获得一个高亲和硝态氮转运蛋白(NRT2)基因,命名为Me NRT2.1,该基因含有1 593 bp的开放阅读框架,编码530个氨基酸。生物信息学分析结果表明,木薯Me NRT2.1与苜蓿、拟南芥、可可、杨树等物种的NRT2.1同源性高,其中与可可树Tc NRT2.1的亲缘关系最近,氨基酸相似性达到90%。实时荧光定量PCR检测结果表明,Me NRT2.1在木薯组培苗的根中表达,并且在NO_3~-浓度为0.2 mmol/L时,其相对表达量较高,NO_3~-浓度为10 mmol/L时其相对表达量较低,NO_3~-浓度为0时几乎不表达;Me NRT2.1在茎、叶中也几乎不表达,即该基因具有诱导型组织特异性表达模式。原生质体瞬时表达发现Me NRT2.1定位在细胞膜上。此研究为进一步通过NRT2基因提高木薯的抗逆性奠定了基础。     

Responses of cassava (Manihot esculenta Crantz) cultivars to in vitro-induced phosphorus stress

Rapid identification of phosphorus (P) stress-tolerant cassava varieties by in vitro and molecular screening is essential for sustainable food production. The objectives of this study were to describe morphological and molecular responses of 10 cassava cultivars to in vitro-induced P stress and identify P-stress-tolerant ones. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing either P or no P (control). The ?tudу was conducted as a 2 × 10 factorial arrangement using a randomized complete-block design with thrее rерliсаtе?. On P-stress medium, TMS 01/1371 did not produce roots, TMS 94/0039 produced roots after three weeks, and MM 96/1751, MM 98/3437, TMS 01/0040, TMS 30040, TMS 84/00353, TMS 91934, TMS 92B/00061, and TMS 94/0026 produced roots after six weeks. Phosphorus-stress treatment reduced number of leaves, number of roots, and number of shoots by 41.8%, 214.7%, 139.2%, respectively. Similarly, P-stress treatment decreased dry weight (DW) of TMS 01/0040, TMS 01/1371, TMS 84/00353, TMS 91934, TMS 92B/00061, and TMS94/0026 by an average of 10.2%. However, P-stress treatment increased DW of MM 96/1751, MM 98/3437 and TMS 94/0039 by an average of 8.4%; P-stress treatment had no effect on DW of TMS 30040. Expression of MeWRKY gene was higher in roots than in leaves of all cultivars by an average of 88.1%. Stress tolerance index relative to P showed that MM 96/1751, MM 98/3437, TMS 94/0039, and TMS 30040 were P-stress tolerant and therefore recommended for breeding and for production to farmers.     

华南6号木薯的选育

华南6号木薯（Manihot esculenta Crantz,cv,M.SC6)是从OMR33-10品系自然杂交种F1无性系后代中选育而成的。其块根产量约35000kg/hm^2，比现推广品种华南205增产50%，比华南201增产48%；块根干物率为40.5%，淀粉质量分数为32.4%，分别比华南205高2.5%和2.3%。     

Responses of cassava (Manihot esculenta Crantz) varieties to in vitro mannitol-induced drought stress

Rapid selection of drought-tolerant cassava varieties by in vitro screening is essential for efficient breeding for drought tolerance. The objectives of this study were to describe the morphological response of 12 cassava cultivars to in vitro mannitol-induced drought stress and identify tolerant ones. Plantlets were raised from nodal segments on Murashige and Skoog (MS) medium containing either 0 g/L or 20 g/L mannitol. Mannitol increased the number of shoots of plantlets per explant (NSS) of all cultivars by an average of 18.2%. However, mannitol decreased the dry shoot weight (DSW) of all cultivars by half. Mannitol decreased total number of leaves (TNL) of plantlets by more than half in all cultivars, except three, in which mannitol had no effect on the TNL. The average root length (ARL) of each one of the following four varieties: TMS 30572, TMS 50395, MM 96/1751, and TMS 94/0026, was not sensitive to mannitol treatment. However, mannitol increased the ARL of MM 98/3437, TMS 94/0039, TMS 92B/00061, TMS 91934 and TMS 84/00353 but decreased the ARL of TMS 05/1654, TME 419, and TMS 92/0326. Mannitol had no effect on dry root weight (DRW) of plantlets of TMS 91934, TMS 94/0026, TME 419, TMS 30572, TMS 50395, and MM 98/3437. The DRW of each of the following four varieties: TMS 05/1654, TMS 92/0326, TMS 92B/00061, and TMS 94/0039, was decreased by mannitol, whereas the DRW of each of TMS 84/00353 and MM 96/1751 was increased by an average of 42.4%. Stress-tolerance index showed that TMS 84/00353, MM 96/1751, MM 98/3437, and TMS 94/0026 were tolerant of mannitol-induced drought stress and, therefore, recommended for planting in drought-prone regions by farmers.     

Identification of cassava (Manihot esculenta Crantz) genotypes with early storage root bulking

Early bulking (EB) cassava varieties shorten the growth period from planting to harvesting. Cassava is preferred by farmers to many other crops because of its high-yielding potential and ability to survive a 4- to 6-month dry season. However, the crop in some cases is only able to remain alive during the dry season without further starch accumulation. This and some other factors, such as pest destruction during dry season and inability to use the same land for cultivation of other crops such as vegetables, therefore necessitate the development of EB cassava genotypes. The objective of this study was therefore to identify EB cassava genotypes using the difference in fresh storage root yield (FSRY) at different plant ages. Thirty-four cassava genotypes were evaluated at Umudike and Otobi in Nigeria in two seasons at two plant ages of 7 and 12 months after planting (MAP) for FSRY. The data generated were used to classify the cassava genotypes as EB or late bulking (LB). Cassava genotypes with less than 100% increase in FSRY at 12 MAP over the yield at 7 MAP were regarded as EB, whereas those with more than 100% increase were regarded as LB genotypes. This basis was used considering the fact that root thickening in cassava commences at about 2 MAP; hence, the root yield at 7 and 12 MAP represents accumulation of assimilates for 5 and 10 months, respectively. About 65%, 62%, and 79% of the genotypes were classified as EB genotypes at Otobi, Umudike, and across the two locations, respectively.