甘蔗基因克隆研究进展

甘蔗是重要的糖料和能源作物,在我国,其糖产量占全国食糖产量的94%。甘蔗是高光合效率C4作物,其蔗糖含量是所有植物中最高的,其基因组中蕴藏着大量重要基因。因此,甘蔗基因克隆对甘蔗品种定向改良具有重要意义。从蔗糖代谢、生长发育和抗逆性三个方面全面总结甘蔗基因克隆的研究进展,并提出今后研究的建议,以期为今后甘蔗基因研究提供参考。     

赤霉素对甘蔗生长的影响

赤霉素对甘蔗生长发育及其产量的效应,正如和其他作物一样表現极为复杂。从我們的試驗結果也可初步看出,赤霉素虽有促进甘蔗生长、增粗、叶面积加大和糖含量提高等作用,但也有抑制分蘗(尤其是前期分蘗)的作用。这种促进和抑制作用,对甘蔗生产来讲都是有利的。因为前者能提高甘蔗的产量和品质。后者,能减少后期的无效分蘗和促进前期分蘗的成林率,避免养分过多的消耗,从而也間接的有利于产量和品质的提高。此外,赤霉素能提高糖含量的結論,与国外报导有矛盾。因此,有必要进一步研究其药效和在生产上推广应用等問題。     

甘蔗非生物胁迫抗性研究进展

甘蔗是世界上重要的糖料和能源作物,对于我国食糖产业发展具有举足轻重的作用。但是,我国甘蔗种植面积正不断减少,呈现出向高海拔、土壤贫瘠等生产条件差的地方转移的趋势,因此遭受的逆境胁迫程度日益加深,严重影响了甘蔗的生长发育及产量形成。如何解决逆境胁迫下甘蔗的产量问题是目前生产上面临的重要课题。目前最好的解决办法还是培育高抗逆品种,为了给甘蔗抗性品种选育提供参考,本文对甘蔗的各种逆境,如低温、干旱、高盐、重金属等伤害与抗逆性的生理生化机制及甘蔗抗逆相关功能基因的挖掘研究进行了综述,以期系统地了解甘蔗逆境研究现状,并提出了甘蔗抗逆育种需要开展的关键工作,以期为甘蔗抗逆相关研究方向的设定、抗性机理和抗性品种的选育提供借鉴。     

甘蔗分蘖发生及成茎的调控研究进展

甘蔗是以收获地上茎为主的重要糖料作物,蔗糖分储藏在蔗茎节间,而分蘖是甘蔗有效茎形成的关键,因此促进甘蔗分蘖成茎是提高甘蔗产量的最有效途径之一。目前,水稻分蘖机理的研究取得了突破性进展,甘蔗也具有禾本科植物特殊的分蘖特性,但相关研究相对滞后,尤其是分子调控机制。本文详细阐述了甘蔗分蘖的生物学特性及现实意义,从栽培技术与管理、环境条件、植物生长调节剂(植物激素)和遗传因素等方面阐述甘蔗分蘖发生及其生长发育的研究结果,为深入研究甘蔗分蘖调控的分子机理提供新视角,也为甘蔗高产栽培技术及分子辅助育种提供理论依据。     

高质量甘蔗基因组DNA的简便快速提取方法研究

甘蔗是世界上重要的糖料作物和能源作物。目前,甘蔗分子生物学研究已成为甘蔗研究的热点之一。基因组DNA的提取是进行甘蔗分子生物学研究的基础。本研究设计含一系列SDS浓度的提取液,同时设加液氮和不加液氮研磨的对比试验,提取甘蔗不同部位叶片的基因组DNA并进行产量和纯度检测以及分子生物学分析。结果表明,所有提取液提取的甘蔗基因组DNA纯度均很高,A260/A280在1.8-2.0之间,A260/A230大于2,但提取液I(0.75%SDS)提取的甘蔗基因组DNA产量较低;加液氮与否对甘蔗基因组DNA的提取产量和纯度没有影响;以提取的甘蔗基因组DNA为模板,分别用一对扩增SPS(蔗糖磷酸合成酶)基因部分片段的引物和一对ISSR引物进行PCR扩增,所有DNA均能扩增出预期的条带;用不同的限制性内切酶对所提取的甘蔗基因组DNA进行酶切,所有DNA样品均能完全酶切。本研究得出最佳甘蔗基因组DNA提取方法如下:磨碎甘蔗叶片后,加DNA提取液(SDS:1.5%;Tris:100 mM;EDTA:20 mM;NaCl:500 mM)于65℃裂解30 min,经酚∶氯仿和氯仿各抽提一次,可获得高产量高质量的甘蔗基因组DNA,能满足后续分子生物学研究的要求。     

甘蔗叶乙酸乙酯部位化学成分研究

甘蔗是糖类加工产业主要的经济作物,甘蔗叶作为广西特色瑶药,在广西民间及瑶族地区具有悠久的药用历史。该课题组前期发现甘蔗叶乙酸乙酯部位具有抗肿瘤活性,为进一步明确其乙酸乙酯部位的化学成分,,该文采用硅胶柱色谱、Sephadex LH-20柱色谱、制备型高效液相色谱等多种分离纯化的方法对甘蔗叶乙酸乙酯部位进行研究。结果表明:从甘蔗叶乙酸乙酯萃取部位分离且鉴定了20个化合物,分别为原儿茶醛(1)、3,4-二羟基-苯甲酸甲酯(2)、3, 4-二羟基苯甲酸(3)、3-羟基-4-甲氧基苯甲酸(4)、对羟基苯甲酸(5)、对羟基苯甲醛(6)、对羟基肉桂酸(7)、丁香酸(8)、3, 5-二甲氧基对苯二酚(9)、1-hydroxy-benzoyl-4-O-α-L-rhamnopyranoside(10)、对羟基苯甲酸-β-D-吡喃葡萄糖酯苷(11)、槲皮素(12)、小麦黄素(13)、异柽柳素(14)、异鼠李素(15)、5, 3'', 4''-三羟基-7-甲氧基二氢黄酮(16)、7-O-甲基圣草酚(17)、[(E)-4-(1S,3R,4R)-1-hydroxy-4,5,5-trimethyl-7-oxabicyclo [4.1.0]heptan-1-yl]but-1-en-3-o-ne(18)、blumenol A(19)和胸腺嘧啶脱氧核苷(20)。其中,化合物1-4、6、9-11、13-16、18和20均为首次从甘蔗叶中分离得到。该研究结果为日后甘蔗叶乙酸乙酯部分的进一步开发提供了依据     

甘蔗杂交品种核心种质重要农艺性状评价及亲缘关系分析

以107份甘蔗杂交品种核心种质为研究材料,ROC22为对照品种,通过新植和宿根2年的田间试验,对蔗茎公顷产量、甘蔗蔗糖分、公顷含糖量等性状进行最小显著差数法(LSD)两两比较和Duncan多重比较评价,筛选优异材料,利用SSR分子标记建立优异材料与我国骨干亲本的亲缘关系。结果表明36份材料在蔗茎公顷产量、甘蔗蔗糖分、公顷含糖量等指标上优于对照品种,可作为亲本进行杂交,且蔗茎公顷产量对公顷含糖量的影响远大于甘蔗蔗糖分对公顷含糖量的影响;20对SSR引物扩增得到292个标记,其中283个为多态性标记;优异材料与我国骨干亲本相似性系数范围在0.384~0.590之间,平均为0.437,存在较大的遗传差异;UPGMA聚类可将所有材料划分为5个类群,蔗茎公顷产量和公顷含糖量表现优异的材料在各类群都有分布,甘蔗蔗糖分表现优异的材料主要集中在Ⅱ、Ⅲ、Ⅳ类群,Ⅲ类群更集中;本研究为甘蔗种质创新、遗传育种提供了优异亲本材料,为杂交组合的配制提供重要指导。     

甘蔗和玉米挥发物差异及其对亚洲玉米螟幼虫取食行为的调控作用

目的】植物挥发物在昆虫行为中发挥着重要的作用,通过研究甘蔗Saccharum officinarum套作玉米Zea mays系统中植物挥发物对亚洲玉米螟Ostrinia furnacalis(Guenée)幼虫取食行为的影响,将为利用化学生态手段防治亚洲玉米螟提供依据。【方法】通过气相色谱-质谱联用仪(GC-MS)分析了甘蔗分蘖期、玉米拔节期的叶片挥发物,并利用行为实验测定了20种化合物(5×10~(﹣3)、5×10~(﹣4)、5×10~(﹣5)、5×10~(﹣6) g/m L)对亚洲玉米螟幼虫取食行为的影响。【结果】甘蔗和玉米的叶片挥发物成分存在差异,甘蔗分蘖期、玉米拔节期叶片中依次分离出49种、52种化合物,其中在甘蔗和玉米叶片中均存在的化合物有43种,甘蔗特有化合物有6种,而玉米特有化合物有9种。甘蔗和玉米的挥发物种类主要包括绿叶气味、萜类和脂肪族化合物(醛类、酮类、酸类、醇类、酯类、酰胺类和烃类),且这些化合物的含量在甘蔗和玉米之间存在着差异,但均以烃类化合物的含量最高。顺-罗勒烯、癸酸、二十七烷、二十九烷对初孵幼虫取食表现引诱活性,壬醛、壬酸、十四烷酸异丙酯、十六烷酸甲酯、二十四烷、二十八烷对初孵幼虫取食行为具有驱避作用。【结论】甘蔗和玉米的叶片挥发物成分存在差异,差异的化合物对亚洲玉米螟初孵幼虫的取食行为具有调控作用。     

甘蔗全长丙酮酸磷酸二激酶（PPDK）基因的克隆

和C3植物相比,C4植物具有明显的生长优势及水分和营养利用率,生物产量也较高。甘蔗是典型的C4作物之一。以甘蔗叶片提取的基因组DNA为模板,以GenBank公布的甘蔗PPDK基因cDNA序列设计引物,进行LA-PCR(Long Acute PCR)扩增。将PCR产物克隆到pMD18-T载体中,转化大肠杆菌JM109,测序,得到了13.5Kb的甘蔗全长PPDK基因序列。为方便后续实验,在引物中引入可利用的XhoI和NotI酶切位点,将全长PPDK基因分两段克隆到pMD18-T Simple载体中,转化大肠杆菌JM109,完成了甘蔗全长丙酮酸磷酸二激酶（PPDK）基因的完整克隆,为将其导入C3作物中奠定了研究基础,实验室保藏菌种。     

菠萝-甘蔗轮作的土壤生态效应

通过2000～2002年进行菠萝-甘蔗轮作田间试验,研究了菠萝、甘蔗轮作对甘蔗土壤生态的影响。结果表明：菠萝甘蔗轮作田与甘蔗连作田比较,能明显地改善土壤的通气和蓄水性,加速速效性养分的释放;增强土壤酶活性;增加土壤微生物总量(一般好气性细菌、真菌、放线菌);有益的氨化细菌和硝化细菌成倍增加,而无益的厌氧性细菌、反硝化细菌则受到抑制;固氮菌数量小且变化不大。菠萝-甘蔗轮作对甘蔗土壤生态环境的改善其实质在于：菠萝、甘蔗轮作后,由于其作物种类的不同导致土壤结构的改善和土壤微生物种类和数量增多,土壤生物活性增强,并导致土壤速效养分的增加。     

甘蔗雌雄蕊的解剖学研究

甘蔗雄蕊花药的壁由四层细胞构成,药室内壁在花药成熟时才发育。花粉母细胞减数分裂属连续型,四分体正常发育为小孢子,但当开花时,只有少数进一步发育为成熟的花粉粒。甘蔗雌蕊柱头是分枝的,每一柱头毛是由数个细胞质浓密的细胞,呈不规则的纵列而组成。倒生胚珠一个,着生于子房室的内侧;内、外珠被均由两层细胞构成,外珠被上侧未能生长到珠孔,内珠被的内层细胞在胚珠发育中不断增大体积。石蜡制片是以樟油为透明剂的。     

甘蔗ACC氧化酶全长cDNA的克隆及序列分析

ACC氧化酶是高等植物乙烯生物合成途径中的限速酶。根据报道的植物ACC氧化酶基因序列设计特异引物,从甘蔗cDNA文库中克隆到一ACC氧化酶基因片段,命名为GZ-ACO,该基因长792bp,与甘蔗基因组文库中克隆的ACO基因片段仅18个碱基之差。根据该cDNA片段序列,设计两对末端扩增的特异引物,利用RACE-PCR技术,获得GZ-ACO片段的5′端和3′端序列。用VectorNTI7.0软件对三个序列进行拼接和分析,结果得到全长的甘蔗GZ-ACO氧化酶基因。GZ-ACOcDNA核苷酸序列长1307bp,具有一个972bp完整的读码框,启动子ATG位于126bp,终止子TAA位于1097bp,推导编码323个氨基酸。系统进化分析表明,GZ-ACO基因氨基酸序列与其它植物已报道的ACC氧化酶基因具有65%~86%的同源率,且与单子叶禾本科植物首先聚类,其次与单子叶芭蕉科、兰科植物聚类,最后与双子叶植物聚类,与植物形态的系统进化结果一致。该基因已在DDBJ/EMBL/GenBank基因数据库注册,注册号为AY521566。     

甘蔗乙烯合成酶基因家族三个成员的克隆与序列分析

ACC（1-aminocyclopropane-1-carboxylic acid）合成酶是高等植物乙烯生物合成途径中的限速酶.根据已克隆的植物ACS（1-aminocyclopropane-1-carboxylic acid synthase）基因同源序列,设计简并引物,以甘蔗叶片总DNA为模板,通过PCR扩增,得到3条特异性强的扩增片段：Sc-ACS1为1 041 bp、Sc-ACS2为1 345 bp和Sc-ACS3为1 707 bp.将序列在GenBank核酸数据库进行同源性搜索,结果表明,3个片段均为ACS基因,推导编码的蛋白质序列分别包含326、242和310个氨基酸.其中,Sc-A CS1和Sc-ACS3同源性最高,核苷酸序列和蛋白质氨基酸序列分别有98%和96%同源,与禾本科植物玉米Zm ACS6、水稻OS-ACS2、毛竹等ACS基因家族也有很高的同源性,核苷酸序列同源性为88%-98%,蛋白质氨基酸序列同源性为73%-81%.甘蔗Sc-ACS2与水稻OS-ACS5在核苷酸和氨基酸序列上分别有91%和79%同源性,但与甘蔗Sc-ACS1和Sc-ACS3基因成员之间,氨基酸同源性分别只有45%和49%.系统进化分析表明,Sc-ACS1和Sc-ACS3基因与玉米Zm ACS6基因亲缘关系最近,而Sc-ACS2基因与水稻OS-ACS5基因亲缘关系最近.Southern杂交表明三基因在基因组中确实存在而且是多拷贝基因.三个片段已在GenBank数据库中注册,注册号分别为AY620985、AY620986和AY788919.     

生长前期叶面喷施乙烯利对甘蔗茎细胞几种酶活性的影响

在甘蔗分蘖初期用乙烯利进行叶面喷施处理 ,并在不同的时期分别对蔗茎细胞质和细胞壁的几种代谢关键酶活性进行测定 ,结果表明 :适当浓度的乙烯利处理提高了整个生长期甘蔗茎细胞质的过氧化物酶活性、生长前中期甘蔗茎细胞质和细胞壁的 Ca2 +-ATP酶、细胞质的 Mg2 +-ATP酶活性和生长后期细胞质的多酚氧化酶活性 ,较高浓度的乙烯利处理普遍提高整个生长期甘蔗茎细胞质和细胞壁的 Ca2 +-ATP酶活性、细胞壁的 Mg2 +-ATP酶活性和旺盛生长期间细胞壁的过氧化物酶活性     

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.     

Evolutionary expansion and functional divergence of sugar transporters in Saccharum (S. spontaneum and S. officinarum)

The sugar transporter (ST) family is considered to be the most important gene family for sugar accumulation, but limited information about the ST family in the important sugar-yielding crop Saccharum is available due to its complex genetic background. Here, 105 ST genes were identified and clustered into eight subfamilies in Saccharum spontaneum. Comparative genomics revealed that tandem duplication events contributed to ST gene expansions of two subfamilies, PLT and STP, in S. spontaneum, indicating an early evolutionary step towards high sugar content in Saccharum. The analyses of expression patterns were based on four large datasets with a total of 226 RNA sequencing samples from S. spontaneum and Saccharum officinarum. The results clearly demonstrated 50 ST genes had different spatiotemporal expression patterns in leaf tissues, 10 STs were specifically expressed in the stem, and 10 STs responded to the diurnal rhythm. Heterologous expression experiments in the defective yeast strain EBY.VW4000 indicated STP13, pGlcT2, VGT3, and TMT4 are the STs with most affinity for glucose/fructose and SUT1_T1 has the highest affinity to sucrose. Furthermore, metabolomics analysis suggested STP7 is a sugar starvation-induced gene and STP13 has a function in retrieving sugar in senescent tissues. PLT11, PLT11_T1, TMT3, and TMT4 contributed to breaking the limitations of the storage sink. SUT1, SUT1_T1, PLT11, TMT4, pGlcT2, and VGT3 responded for different functions in these two Saccharum species. This study demonstrated the evolutionary expansion and functional divergence of the ST gene family and will enable the further investigation of the molecular mechanism of sugar metabolism in Saccharum.     

甘蔗不同部位的固氮酶活性检测

比较巴西2个固氮甘蔗品种‘B1’、‘B8’的叶片、主脉、叶鞘、茎、根和根际周围固氮酶活性的结果表明:茎、根、根际周围的固氮酶活性较高。     

Variations in random amplified polymorphic DNA profile and toxin production among isolates of Colletotrichum falcatum Went from sugarcane in Tamil Nadu,India

Red rot, caused by Colletotrichum falcatum Went, is one of the most important diseases of sugarcane (Saccharum officinarum L.). The pathogen shows a great diversity in virulence as a number of pathotypes are known to occur in nature. In the present study, the toxin producing ability and genetic variability among isolates of C. falcatum collected from major sugarcane growing areas of Tamil Nadu, India were analysed. The C. falcatum isolates differed significantly in their ability to produce toxin in vitro. The toxin from C. falcatum isolate Cf 671a induced the maximum electrolyte leakage (300 μS) from sugarcane leaf tissues. The genetic relatedness of the isolates of C. falcatum differing in toxin production potential was investigated by using RAPD analysis. Analysis of the genetic coefficient matrix derived from the scores of RAPD profiles showed that minimum and maximum percent similarities among the tested C. falcatum isolates were in the range of 19 to 95% respectively. The phylogenetic analysis by the UPGMA identified two main clusters. Cluster A contains only one isolate (Cf 98061) and all the other isolates were placed in Cluster B confirming high genetic diversity among the isolates. No correlation was observed between clustering of the C. falcatum isolates in the dendrogram and their toxin producing abilities.     

The Response of Sugarcane (Saccharum officinarum) Cultured Cells to Anoxia and the Selection of a Tolerant Cell Line

The effect of anoxia on the sugarcane (Saccharum officinarum L.) cultured cells was studied in order to elaborate a technique for in vitro selection of cell lines, which would be tolerant to anaerobic stress. Inhibitory and lethal doses of anaerobic incubation were established from the state of the mitochondrial ultrastructure during the anaerobic incubation of cells either with or without exogenous glucose, as well as from the pattern of the post-anaerobic callus growth. An intact state of the mitochondrial ultrastructure and the viability of some cells in the presence of 3% glucose were shown to be maintained for at least 14 days of anaerobic incubation, while the index of post-anaerobic growth decreased by almost 50% even after 72-hour-long anaerobiosis. In the absence of exogenous glucose, a marked destruction of mitochondria and a twofold decrease in the callus growth index were observed as early as after six-hour-long anaerobic stress. A 48-hour-long incubation under these conditions resulted in the maintenance of the intact ultrastructure only in 7–10% of cells, while a 96-hour-long anaerobiosis brought about the complete degradation of the subcellular structure and cell death. A 48-hour-long anaerobiosis without exogenous glucose was chosen for selecting the anoxia-tolerant cell lines. After three cycles of selection, the anoxia tolerance of the selected cell line exceeded the respective index of the initial callus several-fold. In selected line, about 50% of cells retained viability and could resume growth even after 96-hour-long anaerobic incubation. The experimental results obtained were used to determine the possible causes of the heterogeneity of callus cells as regards their anoxia resistance.     

Direct N2O emission factors for synthetic N‐fertilizer and organic residues applied on sugarcane for bioethanol production in Central‐Southern Brazil

The production and use of biofuels have increased rapidly in recent decades. Bioethanol derived from sugarcane has become a promising alternative to fossil fuel for use in automotive vehicles. The ‘savings’ calculated from the carbon footprint of this energy source still generates many questions related to nitrous oxide (N₂O) emissions from sugarcane cultivation. We quantified N₂O emissions from soil covered with different amounts of sugarcane straw and determined the direct N₂O emission factors of nitrogen fertilizers (applied at the planting furrows and in the topdressing) and the by‐products of sugarcane processing (filter cake and vinasse) applied to sugarcane fields. The results showed that the presence of different amounts of sugarcane straw did not change N₂O emissions relative to bare soil (control). N‐fertilizer increased N₂O emissions from the soil, especially when urea was used, both at the planting furrow (plant cane) and during the regrowth process (ratoon cane) in relation to ammonium nitrate. The emission factor for N‐fertilizer was 0.46 ± 0.33%. The field application of filter cake and vinasse favored N₂O emissions from the soil, the emission factor for vinasse was 0.65 ± 0.29%, while filter cake had a lower emission factor of 0.13 ± 0.04%. The experimentally obtained N₂O emission factors associated with sugarcane cultivation, specific to the major sugarcane production region of the Brazil, were lower than those considered by the IPCC. Thus, the results of this study should contribute to bioethanol carbon footprint calculations.