矮凤梨短营养生长期突变材料‘14-1’的性状鉴定

菠萝栽培种(Ananas comosus var. comosus)的营养生长期较长,制约了其新品种选育效率。收集和筛选营养生长期较短的种质,可为短营养生长期菠萝新品种的选育创制中间材料。从境外收集了23份易成花的野生种质材料,自2008年起进行栽培、繁殖和性状鉴定。通过对营养生长期的比较和观察,从矮凤梨(A. comosus var. nanus)的体胚苗中发现了1份营养生长期显著缩短的突变材料‘14-1’,其营养生长期约6个月(原品种约12个月),1年开花2次,具有极易成花等特点。‘14-1’无性繁殖后代遗传性状稳定,可作为短营养生长期菠萝育种的亲本资源。     

菠萝DNA的提取及AFLP反应体系的建立

为了应用AFLP分子标记对菠萝种资源进行种质鉴定、分类及亲缘关系等方面的研究,最重要的是要获得高质量的DNA,菠萝叶片革质化程度较高,且纤维、多糖、多酚等次生代谢物质含量较多,严重干扰DNA的抽提或影响其后的AFLP双酶切及其扩增反应,本研究以菠萝叶片为材料,利用改良CTAB法得到了39个高质量的菠萝分类群的DNA基因组,并进行了AFLP分析,得到了条带清晰、多态性好的菠萝品种指纹图谱,这为菠萝基因库的建立、优良品种选育以及亲缘关系的系列研究提供理论依据。     

Pineapple organic acid metabolism and accumulation during fruit development

Developmental changes in pineapple (Ananas Comosus (L.) Merrill) fruit acidity was determined for a ‘Smooth Cayenne’ high acid clone PRI#36-21 and a low acid clone PRI#63-555. The high acid clone gradually increased in fruit acidity from 1.4 meq/100 ml 6 weeks from flowering, and peaked a week before harvest at ca 10 meq/100 ml. In contrast, the low acid clone increased in acidity 6 to 8 weeks after flowering, peaked 15 weeks after flowering at ca. 9 meq per/100 ml and then sharply declined in 2 weeks to 6 meq/100 ml. The increased in total soluble solids (TSS) of the low acid clone began 6 weeks after flowering and for the high acid clone at 12 weeks after flowering. The increase in titratable fruit acidity (TA) paralleled the changes in the citric acid content of both clones. Citric acid content increased from less than 1 mg/g at 6 weeks after flowering to 6 to 7 mg/g, 9 weeks later. The malic acid concentration in both clones varied between 3 and 5 mg/g and showed no marked changes just before harvest. The developmental changes in fruit potassium were significantly correlated with fruit acidity and fruit total soluble solids in both the high and low acid clones. Developmental changes in acid-related enzymatic activities showed an increase in citrate synthase (EC 4.1.3.7) activity that occurred a week before harvest, coincided with the peak in citric acid in the high acid clone. An increase in aconitase (ACO, EC 4.2.1.3) activity was observed just before harvest as the decline in acidity occurred in the low acid clone. The activities of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and malic enzyme (ME, EC 1.1.1.40) did not parallel any changes in fruit acidity. The results indicated that the change in pineapple fruit acidity during development was due to changes in citric acid content. The major difference in acid accumulation occurred in the low acid clone just before harvest when acidity declined by one-third. The activities of citrate synthase and aconitase possibly played a major role in pineapple fruit acidity changes.     

菠萝DNA提取方法的研究

采用石英沙代替液氮,用SDS法和CTAB法提取菠萝的DNA。结果表明,SDS法所提取DNA的平均OD值为1.704,提取率为2.098μg/g;CTAB法所提取DNA的平均OD值为1.498,提取率为1.412μg/g。SDS法和CTAB法所提取的DNA都可用作RAPD分析。     

红苞凤梨AbF3′5′H基因上游调控因子筛选

颜色是观赏植物最重要的性状之一,也是当前观赏园艺育种的一个重要方向。为研究红苞凤梨红蓝色彩的呈现机制,初步分析金边红苞凤梨(Ananas comosus var.bracteatus)不同组织中花青素种类与含量,用AbF3′5′H基因启动子构建诱饵载体,利用酵母单杂交(Y1H)文库筛选其上游调控转录因子;并采用酵母单杂交验证技术,验证筛选出的转录因子与AbF3′5′H启动子的互作关系。结果表明：花青素的种类组成和相对比例决定了组织的颜色性状,AbF3′5′H是花青素合成途径种类分支的关键基因,在花青素种类组成与相对比例调控中具有重要作用;AbBTB/POZ、AbHSP81-1和AbGLOX能够与AbF3′5′H的启动子结合而调控其转录,可能在红苞凤梨花青素合成代谢调控中发挥重要作用。研究结果对于进一步揭示红苞凤梨花青素合成代谢调控机理,研究红苞凤梨呈色机理,培育叶色新品种有着重要的理论和实践意义。     

菠萝生长素极性运输载体基因AcPINs和AcAUXs的分离与表达分析

乙烯已经被广泛应用于菠萝人工催花,但其分子机制不是很清楚。以菠萝(Ananas comosus L.‘Perola’)茎尖为材料,采用RT-PCR结合RACE方法得到3个生长素极性运输输出载体基因(Ac PIN1、Ac PIN2和Ac PIN3)和2个生长素极性运输输入载体基因(Ac AUX1和Ac AUX2)的c DNA及基因组DNA全长。Ac PIN1、Ac PIN2、Ac PIN3、Ac AUX1和Ac AUX2的c DNA全长分别为2 690、2 388、2 057、2 156和1 580 bp,其开放读码框长度分别为1 854、1 917、1 530、1 479和1 392 bp,分别编码617、638、509、492和463个氨基酸;其基因组DNA全长分别为3 602、3 208、4 204、5 457和2 436 bp,从起始密码子到终止密码子的长度分别为3 244、2 780、3 947、5 264和2 321 bp。氨基酸序列多重比对及系统发育树结果显示Ac PINs和Ac AUXs分别属于植物PINs和AUX/LAXs家族。荧光定量PCR结果表明,菠萝茎尖经200和1 200 mg·L-1乙烯利诱导处理后,Ac PINs的表达上调较多,其中Ac PIN2在处理后的早期(1~2 d)和后期(28~37 d)显著上调,另外两个PIN家族基因Ac PIN1和Ac PIN3在处理后的大部分时间都明显提高。Ac AUX1的上调表达量相对较少,且相对表达量显著提高也主要集中在处理初期(1 d)和后期(28~37 d),而Ac AUX2的上调表达则只在处理后的前期(1、2、9 d)。研究结果表明,乙烯利诱导菠萝成花过程中,存在着生长素极性运输,且生长素极性输出在此过程中的作用可能更重要。     

Pineapple heart rot isolates from Ecuador reveal a new genotype of Phytophthora nicotianae

Pineapple heart rot disease, caused by Phytophthora nicotianae (syn. P. parasitica), is responsible for significant annual reductions in crop yield due to plant mortality. In Ecuador, new infections arise during the rainy season and increase production costs due to the need for biocontrol and fungicide applications. Studies of P. nicotianae population structure suggest that certain genetic groups are associated with host genera; however, it is not clear how many host‐specific lineages of the pathogen exist or how they are related. The objectives of this study were to determine the level of genetic variation in the P. nicotianae population causing heart rot disease of pineapple in Ecuador and compare the genotypes found on pineapple to those previously reported from citrus, tobacco and ornamentals. Thirty P. nicotianae isolates collected from infected pineapple leaves from four farms were genotyped using nine simple sequence repeat loci. In addition, the DNA sequences of mitochondrial loci cox2 + spacer and trnG‐rns were analysed. Together, these loci supported a single clonal lineage with two multilocus genotypes differing in a single allele and low mitochondrial diversity. This lineage was distinct but closely related to isolates collected from vegetables and ornamentals in Italy. The results support the hypothesis of host specialization of P. nicotianae in intensive cropping systems and contribute to the understanding of population structure of this important pathogen.     

菠萝AcHMGR基因的克隆与表达分析

以菠萝(Ananas comosus L.cv.Comte de Paris)果实为材料,采用RT-PCR结合RACE方法得到3-羟基-3-甲基戊二酸单酰辅酶A还原酶基因(命名为AcHMGR)的c DNA及基因组DNA全长。AcHMGR的c DNA全长2 407 bp,其开放读码框长度为1 740 bp,编码579个氨基酸;其基因组DNA全长为4 115 bp,从起始密码子到终止密码子的长度为3 764 bp,含有4个外显子和3个内含子。荧光定量PCR结果表明,对照的菠萝果实中,AcHMGR基因表达量在花后0 d最高,从花后30 d开始,其表达量急剧下降,之后维持在一个较低水平直到果实成熟。经20 mg/L N-(2-氯-4-吡啶基)-N'-苯基脲(CPPU)处理后,显著提高了AcHMGR基因在花后30 d果实中的相对表达量,为同期对照的1.8倍;但其他时期的相对表达量与对照相比无明显差异。研究结果表明,AcHMGR基因在菠萝果实早期发育过程中表达量较高,CPPU处理提高了AcHMGR基因的相对表达量并显著提高果实的重量,说明CPPU处理后AcHMGR基因可能在促进果实重量的增加中起着重要作用。     

热脱附-气相色谱/质谱分析菠萝果实香气研究

采用热脱附-气相色谱/质谱(TCT-GC/MS)联用技术,对动态顶空密闭循环吸附捕集的菠萝果皮及果肉的挥发物进行了检测,结果表明:菠萝果皮与果肉的香气中富含酯类化合物、尤其是甲酯类成分,含量最高的均为己酸甲酯、分别达到45.66%和64.45%;果皮挥发物中的2-甲基丁酸甲酯相对含量为8.03%、辛酸甲酯相对含量为6.44%,均比果肉中的含量(分别为6.50%和3.51%)高;果肉挥发物中未检出非酯类成分,而在果皮挥发物中检测到2,5-二乙烯基四氢吡喃(9.07%)和少量甲酸乙酸酐(0.87%).     

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

Summary

An efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.