【作者】 周生茂；

【导师】 曹家树； 韦本辉；

【作者基本信息】 浙江大学 ， 蔬菜学， 2009， 博士

【摘要】 山药（Dioscorea spp．）为薯蓣科（Dioscoreaceae）薯蓣属中多年生缠绕性草质植物，其地下块茎富含糖类和酚类物质，普遍用作粮食、药物和生物质能源的原料。迄今已知约有600个山药物种，其中世界上广泛种植的约有10个，而我国大陆地区仅有田薯（D．alata L．）、普通山药（D．opposite Thunb．）和日本山药（D．japonica Thunb．）等3个。山药地下块茎化学成分的组成和含量因基因型和生长环境而异，所以，收集和分类我国主栽山药品种并阐明它们地下块茎糖类和酚类物质形成机理，对人类获得更多高品质山药产品予以消费具有重要意义。本研究以‘紫小’、‘紫大’、‘白扁’、‘白柱’、‘普通’、‘野生’等六种山药基因型为材料，首先，应用山药形态特征多态性、地下块茎可溶性蛋白多态性和SRAP分子标记多态性对它们进行分类评价；其次，运用生理生化手段对它们地下块茎主要糖类和酚类物质的含量及其形成的主要酶活性进行了测定和比较，同时，利用分子生物学技术从六种基因型地下块茎中分离了编码这些主要酶结构基因的cDNA序列，其中重点对‘紫小’的相关基因全长cDNA序列进行了克隆和描述；最后，运用生理生化手段分析了‘紫小’、‘白扁’和‘普通’分别经0．5、1．0、5．0 mmol·L^-1的SA叶喷后地下块茎主要糖类和酚类物质的含量及其形成的主要酶活性；此外，还初步分析了山药糖类和酚类物质形成的可能关系。通过上述研究，获得了以下主要结果：（1）形态学特征多态性、地下块茎可溶性蛋白多态性和SRAP分子标记多态性等三种方法都能以鉴别率为100%的效果将六种山药基因型完全区分为结果相同的3类；‘紫小’、‘紫大’、‘白扁’、‘白柱’属于山药田薯种（D．alata L．），其中还可分为田薯紫色类型和田薯白色类型，前者包括‘紫小’和‘紫大’，后者包含‘白扁’和‘白柱’；‘普通’和‘野生’分别属于普通山药（D．opposite Thunb．）和日本山药（D．japonica Thunb．）。比较这三种分类方法，它们的多态性比率依次为65．45%、67．70%、89．32%，基因型间的遗传相似性系数为0．63～0．96、0．54～0．94、0．36～0．73，遗传距离为0．04～0．37、0．06～0．46、0．27～0．64，其中4个田薯的平均遗传距离分别是0．11、0．15、0．39，这些结果表明SRAP分子标记对上述六种基因型的分类更精细，更适用于山药种质资源的遗传多样性分析和分类。（2）六种山药基因型地下块茎干物质都一直增加，其中田薯白色类型最早积累，‘普通’最迟；AM、AP、Cel和TSS构成地下块茎DW的50%以上，其中仅AM、AP收获时分别占Dw的28．3%～39．1%、34．7%～43．7%；AM、AP的演进总趋势和DW的一样，但Cel、TSS、Suc、TRS、Glc、Frc、TS表现相反；尽管‘野生’的DW、各种可溶性糖、Cel含量都为最小，但是在DC、TS、AM、AP的含量在盛中期前为最大；‘普通’在盛中期前TS、AM和AP的含量最低，但是收获时最大；田薯除Suc含量一直最大外，其他糖类物质含量介于‘普通’和‘野生’间；与可溶性糖和AM相反，DW、TS、AP和Cel在田薯白色品种中较在紫色品种中有更多的含量。SAI、IAI和NI的活性总趋势都是降低，而SuSy增至盛期最大后再降低；转化酶活性都低于SuSy活性，尤其块茎形成初期后；在整个取样时期里，与SAI相反，NI和IAI的活性在‘普通’和‘野生’中高过在田薯中；SuSy活性在‘野生’中都为最低，虽然在块茎形成初期田薯都大于‘普通’，但‘普通’收获时达最大；与IAI、SuSy相反，SAI、NI在田薯紫色品种中的活性大于田薯白色品种的。AGPase、SSS的活性演进模式在同基因型中相似，即‘普通’的一直递增，而其他基因型的增至块茎形成初期或盛期最大后再减至收获；GBSS、SBE的活性变化总趋势在各基因型中均随生育进程而增加，其中在‘普通’、‘野生’中一直递增，虽然田薯在块茎盛后期前也一直递增，但此后SBE与GBSS不同，表现稍有减少。各时期AGPase、SSS、GBSS、SBE的活性在‘野生’中都是最低，在初期虽然‘普通，/小于‘田薯’，但后期则反之；除SBE外，其他三种酶都是田薯的白色品种大于紫色品种。上述除Cel外的糖类化合物含量和相关酶活性在田薯类型内都是‘紫大’大于‘紫小’和‘白扁’大于‘白柱’。方差分析结果显示上述生理生化指标在种间和田薯类型间大多数时期差异显著，但是在田薯类型内大多数时期不显著。同时，Suc虽然和SuSy及淀粉合成相关的酶活性相关性不显著，但是Suc/TSS、Suc/TRS和这些酶活性的正相关性大部分显著。这些结果表明，六种山药基因型地下块茎干物质主要由AM、AP、Cel和TSS构成；各基因型山药地下块茎转化酶和SuSy的活性都存在，转化酶尤其SAI在块茎形成初期有较高的活性，此后SuSy一直是蔗糖裂解的优势酶而导致地下块茎库力的不同；淀粉的基因型著异主要由AGPase、SSS活性的基因型差异所致；各种糖类物质与相关酶活性密切相关，其中蔗糖通过Suc/TSS、Suc/TRS的形式正调控SuSy和淀粉合成相关酶；根据干物质和淀粉积累情况，各基因型熟期依次为田薯白色品种（‘白扁’和‘白柱’）、田薯紫色品种（‘紫大’和‘紫小’）、‘野生’、‘普通’。（3）设计三对简并引物和一些特异引物，运用传统RT-PCR和RACE技术从六种基因型山药地下块茎中都能克隆到SuSy、AGPase和SSS的同源基因cDNA片段，而且三者在各基因型的长度分别为830 b p、784 bp和1 104 bp，可推定编码276、261和367个氨基酸，分别利用这三类同源基因的cDNA序列片段进行同源性比对和系统聚类分析，结合前面基因型分类结果显示，SuSy、AGPase、SSS同源基因的分子系统进化关系一定程度上能反映了山药六种基因型间亲缘关系的远近和进化的快慢。同时，重点对‘紫小’地下块茎的SuSy同源基因cDNA全长进行克隆和分析，DaSuSy1全长cDNA序列大小为2 673bp，其中最大开放阅读框（ORF）、5’端和3’端的非翻译区分别含有2 445 bp、7bp和221 bp，而且3’端的非翻译区含一个24 nt的Poly（A⁺）尾；最大ORF可编码814个氨基酸，分子量为92．76 kDa，等电点为6．42，含有SuSy和葡糖基转移酶两个保守功能域及两个磷酸化位点，即N端的Ser¹⁰和C端的SNLDRRET⁷⁸¹RR(Ser⁷⁷⁴～Thr⁷⁸¹)。该基因在全长cDNA序列、编码区cDNA序列及其编码氨基酸序列的水平上与GenBank中所选已知物种SuSy基因相应序列的同源性分别达45．3%～71．3%、45，8%～74．8%和50．0%～84．7%，与禾本科植物SuSy基因家族中一些成员亲缘关系最近。利用RT-PCR和Northern杂交技术对‘紫小’的DaSuSy1、DaAGPase1和DaSSS1进行表达分析，三个基因在叶片中均没有表达，但是在地下块茎中均有较高丰度的表达；在茎蔓和根中DaSuSy1有微弱表达，DaAGPase1、DaSSS1则完全不表达；DaAGPase1和DaAGPase1可能在转录后水平上受到调控，而DaSSS1可能主要在转录水上受到调控。（4）用0、0．5、1．0、5．0 mmol·^-1的SA在山药地下块茎形成初期和盛后期对‘紫小’、‘白扁’和‘普通’进行叶喷后，通过对TSS、Suc、Glc、Frc、TS、AM、AP、Cel的含量和SuSy、AGPase、GBSS的活性进行测定，结果显示0．5 mmol·L^-1SA提高了所试山药基因型叶片合成Suc及其通过茎基部的量，从而提高了地下块茎除Cel外的所测生理生化参数的含量，尤其在块茎形成初期叶喷效果明显，5．0mmol·L^-1则反之，而1．0 mmol·L^-1在2个田薯品种中显现抑制，在‘普通’中表现促进；同基因型所测参数年间差异大多数不显著；在各基因型中虽然1．0 mmol·L^-1的相关参数与对照的差异也多不显著，但是0．5、5．0 mmol·L^-1和对照的差异大多数显著，而且同SA浓度基因型间也基本显著。这些结果表明，0．5 mmol·L^-1有利于叶片Suc的合成、转运和在地下块茎中向淀粉转化，5．0 mmol·l^-1则反之，1．0mmol·l^-1的作用因基因型而异；三种SA浓度对相关生理生化参数都起平行协同促进或抑制的作用。SA对地下块茎干物质和淀粉积累的影响是通过凋控叶片合成蔗糖和运输而实现，而且蔗糖作为信号分子以Suc/TSS的形式来协同正调控地下块茎的SuSy、AGPase和GBSS的活性；SA各浓度在不同时间里叶喷对糖类物质形成也存在细微差异，为了有利于山药地下块茎干物质和淀粉的积累，适宜浓度的SA应在块茎形成的初期开始每隔30天左右连续叶面喷施，而且普通山药响应SA施用的剂量要高于田薯。（5）设计一对简并引物和一些特异引物，运用普通PCR、RACE和TAIL-PCR技术，从六种基因型山药地下块茎中都能克隆到PAL同源基因cDNA的中间片段序列，利用这些序列进行同源性比对和系统分析，结合前面基因型分类结果显示，虽然可以将‘野生’、‘普通’和田薯的PAL同源基因区分开来，但是田薯4个品种得不到很好的分类，所以该基因不适合用来评价山药基因型的系统进化。同时，从‘紫小’地下块茎中克隆到一个长度为2 376 bp的PAL基因全长cDNA序列，该序列含有一个1 986 bp的最大ORF，一个28 bp的5’端非翻译区，一个362 bp中含25 nt的Poly（A⁺）尾的3’端非翻译区；最大ORF可推测编码一个包括PAL酶活性中心特征（GTITASGDLVPLSYIA）在内的661个氨基酸的多肽，分子量为71．974 kDa，等电点为6．310；DAPAL1和GenBank中已报道其他物种的PAL在全长cDNA序列、编码区序列及其编码氨基酸序列水平上有较高的同源性，都达到50%以上，而且DaPAL1在核苷酸水平上与双子叶植物的更接近，而在氨基酸水平上与单、双子叶植物的接近度著不多；通过用RT-PCR和Northern杂交对DaPAL1的表达进行分析表明，DaPAL1仅在地下块茎特异表达，而且在块茎形成的不同时期所表达的丰度有差异，在块茎形成的初前期表达量最大，此后表达有递减的趋势，直至收获时表达量又有所增加。各期表达丰度和PAL酶活性及总酚含量有正向协同性，说明DaPAL1可能主要在转录水平上受到调控。（6）设计两对简并引物和一些特异引物，运用传统RT-PCR和RACE技术从六种基因型山药地下块茎中都能克隆到CHS和ANS的同源基因cDNA片段，两者在各基因型的长度分别为800 bp、280bp，可推定编码273、93个氨基酸，分别利用这两类同源基因的cDNA序列片段进行同源性比对和系统聚类分析，与前面基因型分类结果相比显示，尽管CHS同源基因的分子系统进化关系一定程度上能反映了山药六种基因型间亲缘关系，但是4NS同源基因不适合。同时，重点对‘紫小’地下块茎的ANS同源基因cDNA全长进行克隆和分析，DaANS1大小为1 387 bp，最大ORF、5’端和3’端的非翻译区分别由1 077 bp、9 bp和301 bp组成；DaANS1最大ORF可编码358个氨基酸，其分子量和等电点分别为40．4 kDa、5．26，并含有依赖于2-酮戊二酸和Fe²⁺氧化的保守结构域，其中包括与2-酮戊二酸特异结合的精氨酸2个(Arg^295、304)及与Fe²⁺结合的保守组氨酸5个(His^{238、243、249、276、294})和天冬氨酸3个(Asp^{240、260、279})；DaANS1与所选被子植物ANS基因的同源性均远高于与裸子植物的同源性，而且与被子植物中双子叶旋花科植物ANS基因的亲缘关系最近，但仅能将属、种间植物合理分类，可评价属、种间植物的亲缘关系。利用RT-PCR和Northern杂交技术分析DaCHS1和DaANS1在‘紫小’中的表达显示，DaCHS1仅‘紫块茎和根中表达，在地下块茎中各时期表达均较强烈，在盛前期表达丰度最高；DaANS1在‘紫小’各个器官中均有表达，但地下块茎的表达总丰度要比根、地上茎和叶片的要强烈得多；DaCHS1、DaANS1可能在转录水平上受到调控。（7）在整个生长发育过程中，TFD、TPA和TTN是山药地下块茎TPC的主要三大构成，分别是总酚的0．64～0．93、0．10～0．25、0．06～0．22，其中TAN和TFL构成大部分TFD；TAN、TFL在田薯紫色类型中分别占TFD的0．53～0．75、0．13～0．34，占TPC的0．49～0．68、0．11～0．29，而在其他基因型中则分别占TFD的0．02～0．1 8、0．55～0．86，占TPC的0．02～0．12、0．38～0．62；这些酚类化合物总变化趋势与干物质和淀粉变化趋势相反，随生育进程而下降，但是和TAC有密切协同性；田薯紫色类型含有高于其他基因型的TPC、TFD，其次是‘普通’和田薯白色类型，‘野生’最低；尽管田薯紫色类型仍含有最高的TAN，但是其TFL和TPA的含量仅稍大于‘野生’，反而是‘普通’和田薯白色类型有相对较高的TFL和TPA，说明山药地下块茎TPA和TFD及TAN和TFL有着竞争性的积累关系；这些酚类化合物在山药种间和田薯类型间差异也在大多数时期差异显著，但是在田薯类型内多数时期不显著。PAL、ANS的酶活性不但在变化趋势上而且在基因型比较结果上都类似于TPC、TAN。不同浓度SA对‘紫小’、‘白扁’和‘普通’的酚类化合物和相关酶的活性在变化总趋势上没有显著影向，但在叶喷后30天左右对这些生理参数的大小有一定作用，而且对各酚类化合物的含量及PAL和ANS的活性都是平行协同影响，其中0．5 mmol·L^-1SA有促进作用，5．0 mmol·L^-1则反之，而1．0 mmol·L^-1在田薯中显现抑制，在‘普通’中表现促进；‘紫小’、‘白扁’和‘普通’的酚类化合物及其相关酶活性不但年间大多数时期差异显著，而且同处理基因型间的差异都显著，同基因型不同处理间的差异也多显著。同基因型的淀粉与各种酚类物质、PAL和ANS的酶活性极显著负相关，说明酚类物质和淀粉在形成时对蔗糖提供的碳素存在着竞争；酚类物质、PAL和ANS也受到蔗糖的调控，而且蔗糖作为信号分子通过Suc/TSS、Suc/TRS而作用；与SuSy的作用相反，转化酶尤其SAI似乎有利于酚类物质形成；SA处理后山药地下块茎糖类物质和酚类化合物的相互关系进一步说明酚类化合物种间差异是由于叶喷SA影响叶片蔗糖合成和转运所致。更多还原

【Abstract】 Yams （Dioscorea spp.） are twining and herbaceous perennials belonging to Dioscorea genus ofDioscoreaceae family,containing many of both carbohydrates and phenols in underground tubers,and areusually utilized as the materials for the productions of subsistence food,druggery,and biomass energy.Sofar,species of about 600 in genus Dioscorea have been reported,of which about 10 species have beengenerally cultivated in the world and only 3 species as D.alata L.,D.opposite Thunb.,and D.japonicaThunb.in land areas of China.Because phytochemicals in underground yam tubers usually vary in bothconstituents and contents with genotypes and ambient growth conditions,both collections and classificationsof cultivated-mainly yam varieties in China and the elucidations of their biosynthesis mechanisms of maincarbohydrates and phenols in underground tubers will be very important significances in yielding moreproducts with higher quality for mankind’s consumptions.In this present study,six genotypic yams namedrespectively‘Zixiao’,‘Zida’,‘Baibian’,‘Baizhu’,‘Putong’,and‘Yesheng’were studied.Firstly,they weresorted and evaluated in the light of their polymorphisms at the levels of morphological characteristics,soluble proteins in tubers,and SRAP molecular markers.Secondly,both contents of major constituents ofcarbohydrates and phenols and the enzyme activities involved in their biosyntheses were measured byphysiological and biochemical methods,meanwhile,the structural gene cDNA sequences coding for the keyenzymes related to the formation of carbohydrates and phenols were isolated from the underground tubers ofsix genotypes,of which full-length cDNA sequences of the related genes in underground tubers of‘Zixiao’were emphasizedly cloned and characterized.Lastly,both contents of major constituents of carbohydratesand phenols and the enzyme activities involved in their biosyntheses in underground tubers were assayed byphysiological and biochemical methods after three genotypic yams as‘Zixiao’,‘Baibian’,and‘Putong’werefoliarly sprayed with SA at concentrations of 0.5,1.0,5.0 mmol.L^-1,respectively.Additionally,the putativerelationships on the biosyntheses of carbohydrates and phenols in yams were assayed.The results obtainedthrough the aforementioned studies were as follows.（1） Based on polymorphisms at the levels of morphological characteristics,soluble proteins in tubers,and SRAP molecular markers,six genotypes of yams were not only all completely differentiated into threeclasses at identification percentage of 100%,but also the same sorted results were obtained by threeclassification methods.Four genotypic yams of‘Zixiao’,‘Zida’,‘Baibian’,and‘Baizhu’belong to D.alataL.,and was also divided into two types named respectively as purple type of yams comprising‘Zixiao’and‘Zida’,and white type of yams containing‘Baibian’and‘Baizhu’.‘Putong’and‘Yesheng’was attributed to D.opposite Thunb.and D.japonica Thunb.,respectively.By comparisons of three classification methods interms of polymorphisms of morphological characteristics,soluble proteins in tubers,and SRAP molecularmarkers,they had respectively polymorphism ratios of 65.45 %,67.7 %,89.32 % for theabovementioned 6 genotypes of yams,and genetic similarity coefficients of 0.63～0.96,0.54～0.94,0.36～0.73,and genetic distance values of 0.04～0.37,0.06～0.46,0.27～0.64,and average genetic distancevalues of 0.11,0.15,0.39 between 4 genotypes of D.alata L.were obtained,These results indicated thatSRAP molecular marker more contributed to both analysis of genetic diversity and classification for yarngermplasms on account of its more accurate classification to the aforesaid 6 genotypes of yams.（2） Dry matters in underground tubers of six genotypic yams straightly increased during growth anddevelopment,and accumulated earliest in white type ofD.alata L and latest in‘Putong’.AM,AP,Cel,andTSS totally accounted for more than 50% of DW in tubers,and AM and AP were 28.3～39.1%,34.7～43.7% of DW at harvesting,respectively.As opposed to Cel,TSS,Sue,TRS,Glc,Frc,TSP,AM and AP hadgenerally the same trends in contents as DW.Although having the smallest contents in DW,each of assayedsoluble sugars,and Cel,in tubers,‘Yesheng’contained the highest ones of DC,TS,AM,AP before the partof the rapidly-bulking stage.Contents of TS,AM and AP in‘Putong’were the smallest before the part of therapidly-bulking stage,and were the largest at harvesting.Four D.alata L.ranged between‘Putong’and‘Yesheng’in the contents of other saccharides except Suc.As opposed to AM and soluble sugars,DW,TS,AP,and Cel presented more contents in purple varityies ofD.alata L.than in white varityies of D.alata L..In studied yam genotypes,general patterns of SAI,IAI,and NI activities all reduced,while SuSy activitiesdecreased to harvesting after straightly increasing to the high point at rapidly-bulking stage.lnvertase werelower in activities than SuSy,especially at the early stage of tuber bulking.By contrast to SAI,IN and IAIhad higher activities in‘Putong’and‘Yesheng’than in four D.alata L during the sampling times.Ascompared to‘Yesheng’with the lowest SuSy activities,four D.alata L.showed higher activities of SuSythan‘Putong’which had the highest activities at harvesting.SAl and NI other than IAI and SuSy were higheractivities in purple varityies ofD.alata L.than in white varityies ofD.alata L..AGPase and SSS activitiesin the same yam had the similar change patterns,namely,both activities of the other yam genotypes except‘Putong’with straight increases in their activities fell to the harvesting after increasing to the highest pointsat the early or rapidly-bulking stage of tuber formation.GBSS and SBE increased in general change trendsof all checked genotypes during growth and development,and straightly increased in‘Putong’and‘Yesheng’Albeit GBSS and SBE in four D.alata L also straightly enhanced before the end of rapidly-bulking stage,thereafter,changes in activities of SBE other than GBSS reduced.‘Yesheng’had the lowest activities ofAGPase,SSS,GBSS,and SBE,and‘Putong’was lower in activities of these enzymes than four D.alata L atthe early stage and vice versa at the later stage.With the exception of SBE,AGPase,SSS,and GBSS hadhigher activities in purple varityies of D.alata L.than in white varityies of D.alata L..The contents of other saccharide except Cel and the above-mentioned enzyme activities were higher in‘Zida’than in‘Zixiao’,andin‘Baibian’than in‘Baizhu’.ANOVA results showed differences in the aforesaid physiological andbiochemical parameters were significant between yam species and between types of D.alata L at themajorities of times,but insignificant for intra-types of D.alata L.At the same time,Suc contentsinsignificantly correlated with activities of both SuSy and the enzymes related to starch biosynthesis,butcorrelation coefficients of ratios of both Suc/TSS and Suc/TRS with them were significant at majorities oftimes.The above-mentioned results indicated that,dry matters in underground tubers of six yam genotypeswere mainly composed of AM,AP,Cel,and TSS;invertase and SuSy had activities in underground tubers ofeach of yam genotypes at every stage,and invertase especially SAI had higher activities at the early stage oftuber formations,and thereafter SuSy was promidant enzyme which caused different sink sizes of six yamgenotypes;the differences in starch between yams mainly attributed to differences in activities of bothAGPase and SSS;saccharide contents were closely relate to the related enzyme activities,of which Sucpossibly up-regulated both SuSy and the aforesaid and starch-related enzymes in forms of Suc/TSS andSuc/TRS;as to the maturing stage of underground tubers,the orders of six genotypes were white type of D.alata L.（‘Zixiao’and‘Zida’）,purple type of D.alata L.（‘Baibian’and‘Baizhu’）,‘Yesheng’,and‘Putong’.（3） Homologous gene cDNA segments coding for SuSy,AGPase,and SSS were respectively isolatedfrom the underground tubers of six genotypic yams with RT-PCR and RACE techniques,and were 830 bp,784 bp,and 1 104 bp in sizes for putative amino acid sequences of 276,261,and 367,respectively.Theresults of both sirnilarities and systemic analyses of these cDNA segment sequences in combinations of theresults of yam classifications showed that SuSy,AGPase,SSS genes could all used to evaluate therelationship among six yam genotypes.At the same time,SuSy gene complete cDNA sequence was clonedfrom the underground tubers of yam‘Zixiao’.DaSuSyl full-length cDNA sequence was 2 673 bp in size,which comprised a 2 445-bp largest open reading frame （ORF）,a 7-bp 5’noncoding region and a 221-bp 3’noncoding region with a 24-nt poly （A⁺） tailor.A polypeptide of 814 amino acids with a 92.76-kDamolecular weight and a theoretical pI of 6.42 was putatively encoded by the largest ORF,containing bothtwo conserved functional regions as SuSy and glucosyl-transferase and two phosphorylated sites as Ser¹⁰ atN-end of and SNLDRRET⁷⁸¹ RR(Ser⁷⁷⁴～Thr⁷⁸¹) at C-end of amino acid sequence.At the levels of completecDNA sequences,coding regions and its deduced amino acid sequences,DaSuSyl shared the similarities of45.3 %～71.3 %,45.8 %～74.8 %,50.0 %～84.7 %,respectively,with SuSy genes of the selected andknown species in GenBank,and had the closest genetic relationship with some members of SuSy genefamilies of gramineous plants.DaSuSyl expressed only in non-photosynthetic organs of yam was not onlystrongly expressed in the underground tubers,but also its expression abundances progressively increasedfrom the initiation of early stage to the part of middle stage of the underground tuber bulking,and thereaftergradually decreased.Expressions of DaSuSyl,DaAGPasel,and DaSSS1 by RT-PCR and Northern hybridization showed that,three genes did not expressed in leaves of‘Zixiao’,but expressed more highly inunderground tubers;although DaSuSy1 expressed a little in stems and roots,DaAGPasel and DaSSS1 didnot completely;DaAGPase1 and DaAGPasel were possibly regulated after transcription and DaSSS1 atlevels of transcription.（4） After‘Zixiao’,‘Baibian’,and‘Putong’were foliarly sprayed with salicylic acid （SA） at the levels of0、0.5、1.0、5.0 mmol.L^-1 at the earlier stage and the termination of rapidly-bulking stage of the formation ofunderground yam tubers,respectively,both contents ofTSS,Suc,Glc,Frc,TS,AM,AP,Cel and activities ofSuSy,AGPase,GBSS were assayed.The results showed that,as opposed to5.0 mmol.L^-1 SA,0.5mmol.L^-1could enhance amounts of both Suc synthesized in leaves and its mobilization through stem base ofthe studied yam genotypes,and further increased contents of the physiological and biochemical parametersassayed,especially,these effects showed evidently on spraying at the earlier stage of tuber formations,while1.0 mmol.L^-1 had negative effects on two cultivars of D.alata L.and positive actions on‘Putong’;differences in the majorities of the assayed parameters in the same yam genotypes were insignificantbetween years;although the aforesaid parameters also bulkly had insignificant differences between 1.0mmol.L^-1 and the control,but were usually significant between 0.5 mmol.L^-1 or 5.0 mmol.L^-1 and the control.These results indicated that,0.5 mmol.L^-1SA contributed to Suc biosynthesis in leaves and its transport andits convesion into starch in underground tubers as opposed to 5.0 mmol·L^-1,wherea effects of 1.0mmol.L^-1varied with genotypes;SA at three levels had paralelled and consistent positive or negative actionson the assayed parameters;the effects of SA on accumulations of dry matters and starch were realized by theregulations of SA to Suc biosynthesis in leaves and transport in stem,and Suc as signal molecularconsistently up-regulated activities of SuSy,AGPase,and GBSS in the forms of Suc/TSS;because SA ofeach concentrations sprayed at different times produced slight and different influences,SA of proper levelsshould be continuously and foliarly sprayed once at every about 30 days from the early stage of tuberformations in order to facilitate accumulations of dry matters and starch in underground tubers,and also D.opposite Thunb.could endure higher levels of SA than D.alata L.（5） Partial cDNA of PAL homologous genes were amplified from underground tubers of six genotypicyams using traditional PCR,RACE-PCR and TAIL-PCR with both a pairs of degenerate primers and somespecific primers.The results of both similarities and systemic analyses of these cDNA segment sequences incombinations of the results of yam classifications showed that,although‘Putong’,‘Yesheng’,and D.alata L.were differentiated in items of PAL homologous genes partial sequences,four cultivars ofD.alata L did not,so PAL gene was not suitable to evaluate phylogeny of yam genotypes.Meantime,A 2 376-bp full-lengthcDNA of PAL gene were isolated from the tubers of‘zixiao’,which contains a 1 986-bp ORF,a 28-bp5’UTR and a 362-bp 3’UTR with a 25-bp ploy （A⁺） tailor.The ORF encodes a 661 AA putative peptide withthe MW of 71.974 kDa and a pl of 6.310.Sequence characters for enzyme active site （GTITASGDLVPLSYIA） was observed in the amino acid sequence.DaPAL1 shared over 50 % similaritywith its homologous genes in other species at the levels of complete cDNA sequences,coding regions and itsdeduced amino acid sequences.DaPAL1 were closer to dicot PAL at the nucleotide sequences,and equallycloser to dicot or monocot PAL at amino acid sequences.RT-PCR and Northern blot analysis showed thatDaPAL1 was expressed only in the tubers and its abundance fluctuates in different stages of the life span.Amaximum expression of DaPAL1 was observed at the initiation of the early stage of tuber formation,thereafter,DaPAL1 expression decreased before the harvesting with slight increase.DaPAL1 expression andits enzyme activity and TPC had the same change patterns,which indicated that DaPAL1 was possiblyregulated at transcription level.（6） Homologous genes cDNA segments coding for CHS and ANS were isolated from the undergroundtubers of six genotypic yams with RT-PCR and RACE techniques,which were 800 bp,280 bp in sizes forputative amino acid sequences of 273,93,respectively.The results of both similarities and systemic analysesof these cDNA segment sequences in combinations of the results of yam classifications showed that,although CHS genes could used to evaluate the relationship among six yam genotypes,ANS genes could not.At the same time,ANS gene complete cDNA sequence was cloned from the underground tubers of yam‘Zixiao’,which wasl 387 bp containing a 1 077-bp largest ORF,a 9-bp 5’ noncoding region and a 301-bp 3’noncoding region.DaANS1 could encode a polypeptide of 358 amino acids with a 40.4-kDa molecularweight and a theoretical pI of 5.26,in which had the 2-oxoglutarate- and Fe²⁺-dependent conservedoxidation regions containing two of conserved Arginine (Arg^295,304) related to the combination of2-oxoglutarate and the Fe²⁺ combination-related amino acid residues as both five of conserved Histidine(His^{238,243,249,276,294}) and three of Aspartate (Asp^240,260,279).DaANS1 shared the higher similarities with ANSgenes of the selected angiosperm species than gymnosperm ANS genes at the levels of complete cDNAsequences,coding regions and its deduced amino acid sequences,and had the closest genetic relationshipwith ANS genes of convolvulaceae plants in dicots of angiosperms,but was used to reasonably sort onlybetween genus or species plants.Expressions of DaCHS1 and DaANS1 by RT-PCR and Northernhybridization showed that,DaCHS1 expressed only in both underground tubers and roots,and had highexpression abundance at every stage,of which most strongly expressed at the former part of rapidly-bulkingstage;DaANS1 expressed in all organs,but more strongly expressed in tubers than in the other organs.DaCHS1 and DaANS1 were possibly regulated at levels of transcription.（7） During growth and development,TPC in underground yam tubers was composed of TFD,TPA,andTTN,which were respectively 0.64～0.93,0.10～0.25,0.06～0.22 of TPC.TAN and TFL mainlyconstituting TFD had respectively 0.53～0.75,0.13～0.34 of TFD in underground tubers of purple type of D.alata L.and 0.49～0.68,0.11～0.29 of TPC,while having respectively 0.49～0.68,0.11～0.29 of TFD inthe other yam tubers and 0.02～0.12,0.38～0.62 of TPC.General change trends of these phenol contents reduced with growth and development of tubers as opposed to ones of starch,and closely cooperated withTAC.Purple type of D.alata L.had higher TPC and TFD contents than the other genotypes,followed byones of‘Putong’and white type of D.alata L.,and‘Yesheng’was the lowest contents of TPC and TFD.Albeit having the highest content of TAN,purple type of D.alata L.was only higher contents of TFL andTPA than‘Yesheng’,and lower than‘Putong’and white type of D.alata L.,which indicated that there wascompetitive relationship between TPA and TFD and between TAN and TFL.Significant differences incontents of these phenols between yam species and between types of D.alata L were observed at themajorities of stage,but did not for intra-types.The change trends of PAL and ANS activities were not onlythe same as ones of TPC and TAN,but also the results of comparisons in PAL and ANS activities betweengenotypes were similar with those of TPC and TAN.As compared with the control,general change trends ofphenol contents and the related enzyme activities did not have markedly differences after foliar spray of SAat different levels,their values would have somewhat changes about 30 days after spray,furthermore,SAshowed the synergistic actions on all kinds of phenols and activities of PAL and ANS,namely,0.5 mmol.L^-1SA had positive effects by contrast to 5.0 mmol.L^-1,and 1.0 mmol.L^-1 was positive to D.alata L andnegative to‘Putong’.The aforesaid parameters of‘Zixiao’,‘Baibian,and‘Putong’at the majorities of stageshad significant differences between years,between genotypes,and between treatments.Starch content inunderground tubers had strikingly significant and negative correlations to each of phenol contents,activitiesof both PAL and ANS in the same yam genotype,which phenol biosynthesis competed with starch formationfor requirements of sucrose.Phenol formations,activities of PAL and ANS were regulated by sucrose,andalso sucrose as signal molecular affected them in the forms of Suc/TSS,Suc/TRS.As opposed to SuSy,invertase especially SAI seemed to facilitate phenol biosynthesis,After SA treatment,the interrelationshipsof carbohydrates with phenols in underground yam tubers further indicated that differences in phenolcontents between yam species were caused by the effects of foliar-spray SA on biosynthesis and transport ofsucrose in leaves.更多还原