利用改良RACE方法克隆木薯淀粉合成关键酶AGPase小亚基基因的研究

首次从木薯中分离克隆出AGPase 小亚基的基因5’端cDNA序列,最终为进一步成功获得木薯淀粉合成关键酶AGPase小亚基基因的cDNA全长序列提供依据。本研究以木薯作为试验材料,设计锚定引物,并根据木薯AGPase 小亚基基因已知序列设计PCR特异引物,利用逆转录引物AUP1 反转录总RNA,然后分离纯化反转录产物,通过对三大酶促反应（逆转录反应,TdT 加尾,巢式及降落PCR技术）的具体改良,并将其与传统方法进行比较。利用改良后方法,首次成功获得木薯淀粉合成关键酶AGPase 小亚基基因5’端cDNA序列。同时,本研究通过对RACE技术的改良与传统方法相比,在操作上更加简单、快速;在序列获得上更加高效;在试验成本上更加低廉。本研究可以高效且快速的成功获得木薯淀粉关键酶AGPase小亚基基因5’端序列,具有一定的实用性及简便性。     

转PPase基因对马铃薯块茎休眠相关生理特性的影响

马铃薯块茎的休眠和发芽对于马铃薯栽培、贮藏保鲜和加工等具有十分重要的意义。本研究以25℃室温条件下贮藏90d后的转正义和反义无机焦磷酸酶(PPase)基因马铃薯Favorita的块茎为材料,对贮藏块茎的PPase活性和及Pi、可溶性糖、淀粉、蛋白质含量进行了测定,以探讨PPase基因对马铃薯相关休眠生理特性的影响。结果表明,与对照相比,转正义PPase基因的两个株系F-2-1和F-2-4块茎中的PPase活性、Pi含量和可溶性糖含量增加、淀粉含量和蛋白质含量降低,且与对照相比均达到极显著水平(只有F-2-4淀粉含量与对照相比无显著性变化);转反义PPase基因的两个株系F-1-1和F-1-2块茎中PPase活性、Pi含量和可溶性糖含量降低、淀粉含量增加,与对照相比均达到极显著性水平,而蛋白质含量与对照相比无显著性变化。     

木薯淀粉分支酶SBEⅠ反义基因遗传转化木薯的研究

旨在获得转淀粉分支酶反义SBEⅠ基因的‘华南木薯8号’转基因植株,为利用转基因技术改良木薯淀粉品质打下基础。在建立了木薯从胚状体子叶到完整植株的再生体系的基础上,用块根特异表达启动子Sporamin驱动的木薯淀粉分支酶SBEⅠ反义基因,通过农杆菌介导法对‘华南木薯8号’进行遗传转化。共接种‘华南木薯8号’子叶517块,获得7株生长良好的转化再生植株,转化再生频率达到1.35%。经PCR检测,其中5株转化再生植株扩增出目的条带,初步证实木薯淀粉分支酶SBEⅠ反义基因已整合进了‘华南木薯8号’基因组中。通过农杆菌介导法可以将淀粉分支酶SBEⅠ反义基因导入到‘华南木薯8号’基因组中,获得了5株转基因植株。     

Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato

Summary Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Analysis of antisense RNA mediated inhibition of GBSS gene expression in large numbers of tubers from in vitro grown, greenhouse grown and field grown transgenic potato plants revealed stable and total inhibition of GBSS gene expression in one clone. In three other transgenic genotypes partial and unstable inhibition was found. In these genotypes both GBSS activity and amylose content were remarkably reduced compared with the non-transformed control genotype. No relationship was found between the level of inhibition of GBSS gene expression and yield and dry matter content.     

Further Characterization of rollC Transgenic Tetraploid Potato Clones, and Influence of Daylength and Level of rolC Expression on Yield Parameters

Tetraploid rolC transformants have been analyzed considering the level of rolC expression and yield parameters. In rolC transformants a maximum of five integrated copies of the gene were detected by Southern analysis. A relation between copy number of the rolC gene, phenotypic rolC expression and intensity of morphological alterations was established. Three experiments were performed to assess the differential effect of the rolC gene on yield and its interaction with growth factors. In the first experiment, the influence of day length on plant morphological and physiological characters was studied. The phenotype of the transgenic plants with a low rolC expression was like the control under the two day-regimes tested, while the intermediate expressing genotype showed a different reaction. In the second and third experiment, tuber yield, tuber number and weight per tuber were recorded under short and long day conditions. When grown in smaller pots of 1.15 1 volume, the control plants had the highest yield of tubers under both day lengths tested. Yield decreased with increasing rolC expression. Tuber number revealed to be genotype dependent: it was higher for low rolC expressing plants and lower for intermediate rolC transformants (Experiment 2). In the third experiment the influence of variable pot size and shape was analyzed for one clone with intermediate and two with low level of rolC expression. Remarkable differences between pots and flat boxes of the same volume were noted for tuber yield and number: Under the two conditions the control clone and the low rolC expressing genotypes gave similar tuber yield and number in both growth containers, whereas the rolC intermediate genotype yielded significantly more in flat boxes. The effect of variable hormonal levels induced in transgenic plants through prokaryotic genes are discussed with the existence of non-predictable genotype in relation to environmental interactions. It is concluded that at least the agronomic performance of potatoes transformed with these genes cannot be predicted on the basis of simple linear relationships among the expression of the gene and of the traits.     

不同木薯品种抗衰老生理与淀粉积累特性研究

以新选056、南洋红、GR891、辐选01、华南124和新选048共6个木薯品为材料,于生育后期分析其叶片过氧化物酶活性、丙二醛、可溶性糖、自由水、束缚水含量以及块根淀粉积累等,以探讨木薯衰老生理与块根淀粉积累的关系。研究结果表明,不同木薯品种生育后期抗衰老生理与淀粉积累特性有明显差异,生育后期茎叶生长和生理机能最旺盛的是华南124,最弱的是南洋红,居中的是辐选01、GR891、新选048和新选056。木薯后期茎叶生长状况对块根淀粉积累有明显影响,过早衰老,降低块根淀粉含量及块根产量;过旺使光合产物大量用于地上部分茎叶生长,而运到地下部的相对减少,从而降低块根淀粉积累和块根产量。因此,木薯生育后期茎叶生长的调控,使其保持在适当水平,是提高木薯块根淀粉积累和块根产量的一项重要措施。同时,生育后期抗衰老生理指标也可作为高产和高淀粉含量品种选育的参考。     

导入AGPase基因的转基因可育水稻及其经济性状的研究

以农杆菌介导法将淀粉合成关键酶AGPase(腺嘌呤二磷酸葡萄糖焦磷酸化酶）基因导入云南地方优质水稻合系35，38，39，41，42中，获得了大量的可育转基因植株。PCR扩增检测，外源基因已稳定整合进水稻基因组中。大量的农艺性状及淀粉含量研究表明，转基因水稻种子的千粒重比对照明显提高，但总淀粉含量没有显著变化。     

H2S and COS Gas Exchange of Transgenic Potato Lines with Modified Expression Levels of Enzymes Involved in Sulphur Metabolism

Different transgenic potato lines were generated for improving the nutritional value of tubers by an advanced perception of their sulphur metabolism. So far no data exist about possible implications for plant health and stress resistance. Metabolite analysis revealed that modifications of enzymes involved in sulphur metabolism were necessarily not reflected in distinctly altered thiol contents. The release of H₂S by plants is putatively involved in pathogen resistance, because of its fungi‐toxic mode of action. The emission of H₂S was determined in 16 potato lines with modified expression level in ATP sulphurylase (ATPS), serine acetyltransferase (SAT), O‐acetylserine(thiol)lyase (OASTL), homoserine kinase (HSK) and threonine synthase activities. The emission significantly increased by factor 7 in one of the ATPS antisense lines and by factor 8 in one of the OASTL antisense lines. A strong increase in H₂S emissions was observed in transgenic plants based on the potato cultivar White Lady, which expressed the Escherichia coli SAT. In addition, the exchange of COS was determined in relation to genetic modifications. Generally, plants act as a sink for COS, but all transgenic lines expressing the E. coli HSK and one of the ATPS antisense lines emitted COS indicating to strong changes in the metabolism of these plants. Such alterations in the gas exchange of transgenic potato plants will most likely also affect their resistance against biotic and abiotic stress.     

反义PPO基因对马铃薯块茎褐化的影响

对刚收获和贮藏5个月后的12个转基因马铃薯品系及对照块茎进行褐化指标的生理检测,发现2个检测时期的各褐化指标在供试品系间差异均达到显著或极显著水平。综合2个检测时期并与对照相比,PPO活性降幅为26.01%~48.65%;酚物质含量降低22.83%~54.81%;褐化强度除GD-9-qc-1低于对照40.45%~46.78%外,其余品系高于对照或与对照无差别。切割块茎发现,大部分转基因品系较对照褐化出现晚,褐化程度低,褐化指数低于对照88.0%~98.86%。PPO的化学定位显示,低褐化转基因品系的多酚氧化酶集中于维管束环,含量明显少于对照,其中GD-9-qc-1仅有少量多酚氧化酶显现出来,推测是由于反义PPO基因抑制块茎POT32基因表达的结果。进一步的方差、相关分析表明,除对多酚氧化酶含量抑制外,反义PPO基因还降低酚物质含量以减轻块茎损伤褐化。     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

木薯叶片可溶性糖含量与块根淀粉积累的关系

试验以辐选01，华南5号(SC5)，新选048，新选056，南洋红（SC201），华南205(SC205)，GR891，华南124（SC124）为材料，分别在块根形成期、块根膨大期和工艺成熟期测定叶片可溶性糖含量和块根淀粉含量，探讨木薯叶片可溶性含量与块根淀粉积累的关系。结果表明，从块根形成期到工艺成熟期，叶片可溶性总糖含量与块根淀粉积累都逐渐增加，叶片可溶性总糖含量与块根淀粉积累呈正相关，从块根膨大期到工艺成熟期，叶片还原糖含量逐浙增加，与块根淀粉积累呈正相关，而叶片蔗糖含量逐浙降低。与块根淀粉积累呈负相关。8个品种都有同样的趋势。     

木薯栽培种与野生种叶片光合器官结构和酶学差异

为探究栽培型木薯高光效的机理,采用石蜡切片法和酶活测定等方法分别对木薯栽培品种Arg7(Manihot esculenta)和野生种W14(Manihot esculenta subsp. flabellifolia)进行了叶片解剖结构、光合淀粉累积关键酶活性、净光合效率和叶绿素含量的测定。对比分析两木薯种试验结果,发现栽培型木薯较野生种具有独特的叶片维管束鞘细胞结构,较高的PEPCase(50.41NADHμmol/(mg protein?hr)活性和净光合速率(36.82μmol/(m2s))以及低于野生种的叶绿素含量。另外,木薯种Arg7叶片中Rubisco、PEPC、AGPase和SuSy这4个酶活性动态变化与净光合速率变化趋势基本一致,表明木薯光合作用、淀粉合成以及光合产物的运输是个协同作用的过程。最后推断出栽培型木薯高光效的原因可能是叶片中存在一定程度的C4光合模式以及较野生种通畅的光合产物运输通路。     

Potato germplasm development for warm climates: genetic enhancement of tolerance to heat stress

Complex potato hybrids were derived through unilateral sexual polyploidization between Solanum tuberosum cv. Atlantic and 11 diploid hybrids that produced 2n pollen through co-orientation of second division spindles. The hybrids represented the following genomic compositions: TAPB, TAPC, and TAPM where T = S. tuberosum, A = S. andigena, P = S. phureja, B = S. berthaultii, C = S. chacoense, and M = S. microdontum. The B, C, and M components of the hybrids had been selected from heat tolerant accessions. The heat tolerance and agronomic performance of the hybrids were assessed under both controlled environments and field tests. The hybrids exhibited good tuberization potential (tuber number, tuber weight and percent plants producing tubers) under heat stress conditions in controlled environments. Under severe heat stress conditions in the field in Israel, many of the hybrids tuberized when Atlantic failed to produce any tubers at all. Under more moderate heat stress in Virginia, the complex hybrids exhibited total tuber yield not significantly different from Atlantic, although the tuber set per plant was greater. However, the complex hybrids were more tolerant to heat necrosis and hollow heart than Atlantic. Total glycoalkaloids in field-grown tubers did not exceed the acceptable limit for 13 of 26 selections examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

钾肥对高淀粉马铃薯块茎淀粉合成相关酶活性的影响

以东北地区选育的2个高淀粉马铃薯品种东农310和克新22号为试验材料,研究不同施钾量对高淀粉马铃薯品种块茎形成过程中淀粉合成相关酶活性的影响。结果表明,随着生育期的推进,不同施钾量处理马铃薯品种东农310和克新22号块茎中的腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、颗粒凝结性淀粉合成酶(GBSS)、可溶性淀粉合成酶(SSS)、淀粉分支酶(SBE)都呈现先增加、达到峰值后又开始下降的单峰曲线变化。增施钾肥对东农310和克新22号块茎淀粉合成中AGPase和GBSS活性影响比较大,而对SSS和SBE的活性影响较小。     

Effects of Exogenous Injection of Different Sugars on Leaf Photosynthesis, Dry Matter Production and Adenosine 5'-Diphosphate Glucose Pyrophosphorylase (AGPase) Activity in Sweet Potato, Ipomoea batatas (Lam.)

Solutions of sucrose, glucose and fructose were artificially injected into the stems of sweet potato plants. The effects of solution injection on both dry matter production and the activity of adenosine 5'-diphosphate glucose pyrophosphorylase (AGPase) in tuberous roots were investigated and compared. The total weight of carbon (T_C) artificially and photosynthetically supplied to a plant during the treatment period of 40 days was 0.987–1.869 times the weight of photosynthetically assimilated carbon alone. At the final sampling time, the dry matter weight of tuberous roots in the plants injected with sugar solutions showed a 2.73–9.13-fold increase over that of the control plant. The root weight linearly increased with T_C. The activity of AGPase was also enhanced by solution injections, with 27–63 % increases compared to the control, but was not significantly related to T_C. The injection of sugar solutions is concluded to have a dual effect on root production in sweet potato. One effect is that the increased sugar concentration in the plant increases AGPase and sink activities, and the other effect is that the increased carbon supply quantitatively promotes starch synthesis and accumulation in roots.     

木薯AGPase基因的克隆与载体构建

为了研究木薯淀粉合成酶关键基因AGPase在木薯淀粉合成中的作用,根据GenBank中其他物种AGPase小亚基基因序列,从高淀粉木薯‘辐选01’中克隆得到含3’末端的909 bp的基因片段,回收克隆测序。结果显示,克隆获得片段与基因库中登录的蓖麻、毛果杨、甜橙、番薯、蜜柑、大豆同源性依次达到91%、91%、87%、86%、86%及85%。将阳性克隆与PBI121载体进行双酶切、连接,构建植物反义表达载体PBI121-AGPss,并将其导入农杆菌,得到农杆菌工程菌株。为进一步开展木薯淀粉合成相关的基因调控、转基因及基因功能研究奠定基础。     

5个木薯品种生理特性与主要农艺性状的研究

对木薯品种西选07、桂垦09-11、辐选01、南植199和华南205的生理特性和主要农艺性状进行比较研究。结果表明,5个木薯品种的叶片叶绿素、蔗糖、可溶性糖含量、农艺性状、产量和淀粉含量有显著差异。西选07块根产量最高,其次是桂垦09-11和辐选01;南植199淀粉含量最高,其次是辐选01和西选07;西选07淀粉产量最高,其次是辐选01和桂垦09-11;西选07单株块根数、块根长、块根粗和块根重最高,其次是桂垦09-11和辐选01;南植199叶片蔗糖和可溶性糖含量最高,其次是西选07和辐选01。叶片蔗糖、可溶性糖含量均与块根淀粉含量呈极显著正相关,可作为高淀粉木薯品种评价的生理指标。淀粉产量综合反映木薯淀粉生产能力,可作为木薯品种品质评价的指标。     

转基因纯合四倍体马铃薯多酚氧化酶活性及同工酶谱的变异

选择14个已插入反义PPO基因的纯合四倍体马铃薯“GD-9-qc”系列,进行叶片、微型薯PPO活性检测和同工酶分析,结果表明,不同品系间的叶片、微型薯PPO活性存在极显著差异,其中2个品系的叶片及8个品系的微型薯PPO活性明显低于对照;供试转基因品系间的叶片PPO同工酶谱带的颜色深浅表现差异,所示结果与PPO活性检测基本一致;     

保水剂对土壤理化性状和木薯产量影响研究

为探讨保水剂对土壤理化性状和木薯产量的影响,以木薯品种‘辐选01’为材料,在每公顷施用保水剂(0 kg,20 kg,40 kg)的处理下,研究保水剂不同用量对土壤理化性状、木薯农艺性状及产量的影响。结果表明：施用保水剂能改善土壤理化性状,促进木薯生长,提高木薯产量。其中以40 kg/hm2保水剂施用量效果最好,其土壤含水量、孔隙度、速效氮、速效磷、速效钾分别增加了12.56%、16.47%、11.62%、36.87%、45.42%;株高、茎粗分别增加了14.74%、12.74%;木薯产量和经济收入分别增加了36.96%和22.15%。由此可见,保水剂对改善土壤理化性状,促进木薯生长,增加木薯块根产量,提高经济效益起到积极的促进作用,因此可在木薯栽培上推广应用。     

Influence of the Indoleacetic Acid-Lysine Synthetase Gene (iaaL) of Pseudomonas syringae subsp. savastanoi on Yield Attributes of Potatoes

The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase which conjugates free indoleacetic acid (IAA) with lysine. lAA-lys is biologically less active than free IAA. The iaaL coding region was expressed under the control of the cauliflower mosaic virus 35S promoter and transgenic potato plants were produced (Spena et al. 1991). 35S iaaL potato plants are characterized by increased internodal length and epinastic bending of older leaves. In three greenhouse experiments with plants grown in pots of different size and in two growth chamber experiments tuber number increased in iaaL transgenic plants compared to untransformed and vector-transformed controls of the same genotype. The increase in tuber numbers observed under controlled conditions was reflected in tuber yield which increased in the pot grown transgenics.