日本沼虾三群体线粒体16S rRNA基因片段序列的差异与系统进化

通过对日本沼虾(Macrobrachium nipponense)3个群体线粒体DNA 16S rRNA基因片段进行扩增和测定,得到长度为495bp的片段,其碱基A、T、G和C的平均含量分别为28.6%、36.1%、22.7%和12.5%,AT含量明显高于GC含量。通过对日本沼虾16SrRNA基因片段遗传特征的研究发现其种内变异很小,在3个群体中只有5个位点发生转换。另外,利用其454bp的同源序列,以中国明对虾(Fenneropenaeus chinensis)为外群探讨了沼虾属日本沼虾、罗氏沼虾(M.rosenbergii)等8种沼虾的系统进化关系。用MEGA3.1软件中的NJ法构建的分子进化树,日本沼虾3个群体先聚在一起后与海南沼虾聚在一起;另外,罗氏沼虾与马氏沼虾、短腕沼虾与贪食沼虾亲缘关系较近先聚在一起,然后再与大臂沼虾和等齿沼虾聚在一起,最后才与外群中国明对虾聚在一起。     

日本沼虾ITS1序列分析及SNPs位点的筛选

研究采用直接测序法,分析日本沼虾(Macrobrachium nipponense)rDNA基因内转录间隔区ITS1的DNA序列,以筛选日本沼虾SNPs位点。共分析了32个太湖水域野生日本沼虾样本,结果表明,日本沼虾ITS1序列平均长度为1749.8bp,是迄今已报道的最长的ITS1序列,A、G、T和C的平均含量分别为29.9%、28.3%、27.7%、14.0%,G+C的含量平均为42.3%。通过序列比对,共筛选出22个SNPs位点,SNPs位点出现频率为0.0126,其中9个为C/T转换(占40.91%),4个为A/G转换(占18.18%),2个为A/T颠换(占9.09%),5个为T/G颠换(占22.73%),1个为A/C颠换(占4.55%),1个A/T或C颠换(占4.55%)。日本沼虾ITS1序列的22个SNP位点中,21个位点为2个等位基因,1个位点出现了3个等位基因,为复等位基因位点。日本沼虾ITS1序列中还发现3个具有多态性的微卫星位点、1个高度变异区以及大量的缺失、插入。研究首次对日本沼虾ITS1序列进行了分析,并发现了大量的SNP位点,为日本沼虾遗传育种研究提供了新的分子标记。     

奥利亚罗非鱼与尼罗罗非鱼rDNA内转录间隔区序列特征

核糖体DNA内转录间隔区(internal transcribed spacers,ITS)是经常被用作种和种群水平系统研究的分子序列.本文分离了奥利亚罗非鱼(Oreochromis aureus)、尼罗罗非鱼(O.niloticus)内转录间隔区,包括部分185序列,ITS1、5.8S、ITS2全序列及部分28S序列.4尾奥利亚罗非鱼的10个克隆序列分析表明,其存在长度不同的a、b两种类型ITS1.a型长为536 bp,GC含量为69.96%;b型长为520 bp,GC含量为69.04%～69.42%.4尾尼罗罗非鱼的10个克隆序列分析表明,其只存在a型ITS1,长为536～540 bp,GC含量为69.42%～70.19%.与b型ITS1相比,a型ITS1在16～31 nt有16 bp片段(GGCCCGCCTCGGCGC)的插入.奥利亚罗非鱼和尼罗罗非鱼共20条ITS序列中,5.8S长度均为157 bp,GC含量为56.69%～57.96%;ITS2为408 bp,GC含量为72.79%～74.26%.奥利亚罗非鱼和尼罗罗非鱼ITS区序列相似性高达98.2%,表明这两种罗非鱼亲缘关系很近.此外,本文对14尾奥利亚罗非鱼、15尾尼罗罗非鱼以及15尾奥尼罗非鱼[O.aureus(♂)×O.niloticus(♀)]ITS1的扩增结果显示,奥利亚罗非鱼均有a、b两种类型ITS1;15尾尼罗罗非鱼中1尾为a、b两类型ITS1,14尾为a型ITS1;15尾奥尼罗非鱼中则有6尾具有a、b两类型ITS1,9尾为单一的a型ITS1.分析表明,奥利亚罗非鱼在ITS1这个位点一致性高,但尼罗罗非鱼中有1尾混杂了奥利亚罗非鱼的基因,同时也说明分子生物学手段应用于种质鉴定比形态学手段更为精确.     

罗氏沼虾Rab11基因cDNA克隆及其组织表达分析

为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。     

复方中草药对罗氏沼虾碱性磷酸酶（AKP）和血清蛋白的影响

为了探究中草药添加剂对罗氏沼虾(Macrobrachium rosenbergii)免疫功能的影响,以罗氏沼虾为对象,在水温20～30℃、pH 7～8的试验条件下,往饲料中分别添加质量分数为0%、1.0%、1.5%、2.0%、2.5%、3.0%的复方中草药制剂,连续饲喂25 d。结果显示:随着饲料中复方中草药质量分数的提高,罗氏沼虾的生长指标、血清中碱性磷酸酶(AKP)活性、血清蛋白含量均呈现先上升后下降的趋势,试验组相较于对照组有很大提高;中草药质量分数的提高引起的血清中蛋白含量的提高远远大于肌肉中蛋白的含量,在各试验组中2.5%的复合中草药制剂的添加量效果最好。试验证明,添加复方中草药制剂对罗氏沼虾的免疫活性、生长性能均有明显的促进作用。该结果能够为罗氏沼虾的健康养殖提供基础资料。     

三种沼虾的COI基因序列变异及其分类地位探讨(英文)

罗氏沼虾(Macrobrachium rosenbergii)和日本沼虾(M.nipponense)经在我国得到广泛的养殖,产生巨大的经济效益.祁连沼虾(M. qilianensis)是自然分布在我国甘肃省的土著虾种,因其外部形态符合沼虾属的特征,而被前人归入沼虾属.为了从分子生物学的角度理解罗氏沼虾、日本沼虾与祁连沼虾的遗传差异,为合理开发和利用沼虾资源提供理论基础,作者对这3种沼虾的线粒体COI基因序列进行研究.从甘肃、浙江等地分别采集这三种沼虾的样本各10尾,共30尾,其中祁连沼虾是野生样本,而罗氏沼虾和日本沼虾都是养殖样本.通过PCR方法扩增线粒体COI基因,并测序.通过比对,获得一致序列649 bp.在30个样本中共检测到169个变异位点,占总变异的26.04%;共检测到7种单倍型.3种沼虾的核苷酸多态性分别为:罗氏沼虾0.411%、日本沼虾0.092%、祁连沼虾0.031%.野生的祁连沼虾遗传多样性远远低于养殖的罗氏沼虾和日本沼虾.三种沼虾单倍型之间的Kimura双参数遗传距离在19.87%～23.84%,三者之间的遗传距离较大,提示三者均为有效种.为进一步确定这三种沼虾在长臂虾科的分类地位, 我们从NCBI数据库中下载了长臂虾科的其它种类的COI序列进行系统发生分析.用NJ法构建的分子系统树显示:日本沼虾和罗氏沼虾与沼虾属的其它种类聚成一枝,而祁连沼虾与同亚科的脊尾白虾(Exopalaemon carinicauda)和长角长臂虾(Palaemon debilis)较沼虾属另10种虾的遗传距离近,即祁连沼虾与白虾属及长臂虾属聚成另一枝.凶此,COI序列的结果不支持祁连沼虾归入沼虾属.但其分类地位应该综合多方面证据重新进行分析确定.     

国产“水山姜”的分类学研究—来自核糖体DNA ITS区序列的证据

用直接测序法对国产黑果山姜Alpinia nigra(Gaertn.)Burtt以及“水山姜Alpinia aquatica (Koen.)Rose”。的核糖体DNA中的内转录间隔区（ITS）序列进行了测定,结果显示两者序列完全一致;ITS1长度为178bp,ITS2长度为232bp,5.8S编码区长度为164bp,GC含量为56.9%,形态学特征结合DNA分子证据,认为《中国植物志》记载的水山姜实为黑果山姜。     

不同居群栽培牡丹rDNAITS区序列分析及鉴别

对我国四个地区牡丹主要栽培品种rDNA ITS区序列进行测定,研究各居群rDNA ITS序列特征及差异,建立不同地区主要牡丹栽培品种的区域性分子标记,参照GeneBank信息(登录号:U27692)利用Clustal X、MEGA 3.1分子进化遗传分析软件对测序结果排序.牡丹ITS全序列长度为652 bp,相对于GenBank中牡丹rDNA ITS全序列在448位少一个C碱基,其中ITS1为267 bp,5.8 S为163 bp,ITS2为222 bp,GC含量为55.7%～56.9%,共有30个变异位点,主要发生在ITS1、ITS2区,5.8S区也存在多个位点的变异.牡丹rDNA ITS区序列特征(SNPs)可用于不同地区主要牡丹栽培品种的鉴别.     

日本沼虾含硒谷胱甘肽过氧化物酶全长克隆及表达分析

为探讨日本沼虾(Macrobrachium nipponense)的免疫机制及含硒谷胱甘肽过氧化物酶(Se-GPx)在甲壳动物解毒和免疫应激反应中的作用,本文采用RACE法克隆了日本沼虾Se-GPx cDNA全长,其cDNA全长为908 bp,5′-UTR的长度为91 bp,3′-UTR长为256 bp,包括1个保守的硒代半胱氨酸插入序列(SECIS)和1个polyA尾,开放阅读框长度为561 bp,编码由186个氨基酸组成的多肽,其中,第39个氨基酸为TGA编码的硒代半胱氨酸。同源性和相似性分析显示日本沼虾的Se-GPx在甲壳动物中与罗氏沼虾相似度最高,在脊椎动物中与GPx1和GPx2家族的相似度比较高。日本沼虾Se-GPx基因在血细胞、肝胰腺、肌肉、卵巢、表皮以及大颚器官中都有表达,尤其在血细胞中表达量相对较高。用嗜水气单胞菌(Aeromonas hydrophila)刺激后3h和6h,血细胞Se-GPx基因表达量显著升高。结果表明,日本沼虾Se-GPx作为一种重要的抗氧化酶,在维护组织正常功能方面及免疫应激反应中发挥重要作用。     

三种沼虾的COI基因序列变异及其分类地位探讨

罗氏沼虾(Macrobrachium rosenbergii)和日本沼虾(M. nipponense)已经在我国得到广泛的养殖,产生巨大的经济效益。祁连沼虾（M. qilianensis）是自然分布在我国甘肃省的土著虾种,因其外部形态符合沼虾属的特征,而被前人归入沼虾属。为了从分子生物学的角度理解罗氏沼虾、日本沼虾与祁连沼虾的遗传差异,为合理开发和利用沼虾资源提供理论基础,作者对这3种沼虾的线粒体COI基因序列进行研究。从甘肃、浙江等地分别采集这三种沼虾的样本各10尾,共30尾,其中祁连沼虾是野生样本,而罗氏沼虾和日本沼虾都是养殖样本。通过PCR方法扩增线粒体COI基因,并测序。通过比对,获得一致序列649 bp。在30个样本中共检测到169个变异位点,占总变异的26.04%;共检测到7种单倍型。 3种沼虾的核苷酸多态性分别为：罗氏沼虾0.411%、日本沼虾0.092%、祁连沼虾0.031%。野生的祁连沼虾遗传多样性远远低于养殖的罗氏沼虾和日本沼虾。三种沼虾单倍型之间的Kimura双参数遗传距离在19.87%~23.84%,三者之间的遗传距离较大,提示三者均为有效种。为进一步确定这三种沼虾在长臂虾科的分类地位, 我们从NCBI数据库中下载了长臂虾科的其它种类的COI序列进行系统发生分析。用NJ法构建的分子系统树显示：日本沼虾和罗氏沼虾与沼虾属的其它种类聚成一枝,而祁连沼虾与同亚科的脊尾白虾（Exopalaemon carinicauda）和长角长臂虾（Palaemon debilis）较沼虾属另10种虾的遗传距离近,即祁连沼虾与白虾属及长臂虾属聚成另一枝。因此,COI序列的结果不支持祁连沼虾归入沼虾属。但其分类地位应该综合多方面证据重新进行分析确定。     

Preliminary Analysis of Length and GC Content Variation in the Ribosomal First Internal Transcribed Spacer (ITS1) of Marine Animals

Length and guanine–cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

Ribosomal DNA difference between species A and D of the Anopheles dims complex of mosquitoes from China

Abstract. Species A and D of the Anopheles dints complex were found in China. Ribosomal DNA second internal transcribed spacers (ITS2) of both species A and D were sequenced and found to be 716 and 710 base-pairs in length, respectively, with 699c GC content. No evidence of intraspecific variation was detected in the ITS2 sequence of species A, whereas the sequence of species D showed variation at one position in the ITS2. A large number of simple repeat motifs were dispersed throughout the ITS2 sequences. The level of interspecific difference was 5.4% of the nucleotide sequences. Some of the interspecific differences were located in regions with subrepeat structure.     

Study on Sequences of Ribosomal DNA Internal Transcribed Spacers of Clams Belonging to the Veneridae Family (Mollusca: Bivalvia)

The first and second internal transcribed spacer (ITS1 and ITS2) regions of the ribosomal DNA from four species, Meretrix meretrix L., Cyclina sinensis G., Mercenaria mercenaria L., and Protothaca jedoensis L., belonging to the family Veneridae were amplified by PCR and sequenced. The size of the ITS1 PCR amplification product ranged from 663 bp to 978 bp, with GC contents ranging from 60.78% to 64.97%. The size of the ITS1 sequence ranged from 585 bp to 900 bp, which is the largest range reported thus far in bivalve species, with GC contents ranging from 61.03% to 65.62%. The size of the ITS2 PCR amplification product ranged from 513 bp to 644 bp, with GC contents ranging from 61.29% to 62.73%. The size of the ITS2 sequence ranged from 281 bp to 412 bp, with GC contents ranging from 65.21% to 67.87%. Extensive sequence variation and obvious length polymorphisms were noted for both regions in these species, and sequence similarity of ITS2 was higher than that of ITS1 across species. The complete sequences of 5.8S ribosomal RNA gene were obtained by assembling ITS1 and ITS2 sequences, and the sequence length in all species was 157 bp. The phylogenetic tree of Veneridae clams was reconstructed using ITS2-containing partial sequences of both 5.8S and 28S ribosomal DNA as markers and the corresponding sequence information in Arctica islandica as the outgroup. Tree topologies indicated that P. jedoensis shared a close relationship with M. mercenaria and C. sinensis, a distant relationship with other species.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

栽培种甘薯及其近缘野生种nrDNA ITS序列分析

采用核糖体DNA内转录间隔区(nrDNA ITS)序列比较分析了甘薯及其近缘野生种的遗传多样性及系统进化关系,首次报道了栽培种甘薯‘徐薯18’(Ipomoea batatas‘Xushu18’)及其近缘野生种I.triloba(DOM),I.cordatotriloba(MEX),I.nil(PER),I.nil(JPN),I.hederacea Jacq.(USA),I.hederacea Jacq.(HK)和种间杂交种67-1(I.batatas‘Xushu18’×I.hederacea Jacq.)及回交种(67-1×I.batatas‘Xushu18’)的nrDNA ITS序列。序列分析表明,栽培种甘薯及其近缘野生种nrDNA ITS序列长度为570~600bp。其中,ITS1序列为185~209 bp,GC含量为53.11%~61.83%;ITS2序列为214~226 bp,GC含量为61.21%~72.89%;5.8S序列均为165 bp,GC含量为54.55%~55.76%。此外,栽培种甘薯及其近缘野生种ITS序列信息位点均集中在ITS1和ITS2区;与其他甘薯属植物相比,I.wrightii ITS2的末端缺失了6~8个碱基。系统进化分析表明,栽培种甘薯‘徐薯18’(I.batatas‘Xushu18’)和野生种I.triloba、I.cordatotriloba、I.lacunosa、I.trifida的亲缘关系较近,与I.wrightii、I.pes-tigridis、I.grandifolia、I.nil、I.hederacea Jacq.、I.purpurea的亲缘关系较远;杂交后代与栽培种甘薯‘徐薯18’(I.batatas‘Xushu18’)亲缘关系较近,与野生种父本I.hederacea Jacq.的亲缘关系较远。     

高GC含量的鳜鱼rRNA基因家族的克隆

动物rRNA基因是一种GC含量较高、结构复杂的重复序列。该研究结合亲缘生物法生物信息学技术,经反复摸索后选用LA PCR法即LA PCR Taq酶结合GC缓冲液来扩增鳜鱼复杂的rRNA基因重复序列,经测序鉴定最终克隆了鳜鱼的3个rRNA基因及其2个间隔序列。分析了鳜鱼与相关动物的rRNA基因序列的同源性和进化关系。探索了克隆复杂DNA序列时引物设计的特别规则、反应体系的改进、DNA聚合酶的选用、循环参数的调整等措施。     

Multiple Divergent ITS1 Copies Were Identified in Single Tomato Genome Using DGGE Analysis

The intra-genomic variation in the internal transcribed spacer (ITS) region has led to misleading conclusions in the evolutionary analysis of plants; understanding this variation is critical for correct evolutionary analysis based on ITS sequences. To reveal the ITS variation in tomato, entire copies of ITS1 sequences within tomato species were separated using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. ITS1 copies varied significantly in sequence composition, but not in sequence length within the same tomato cultivar. DNA sequence similarity of the ITS1 copies was 77–100 %. Moreover, AT and GC contents in ITS1 copies from each tomato cultivar were significantly different, ranging from 50.4 to 64.3 % for GC and from 35.7 to 49.6 % for AT. However, the length variation of ITS1 was insignificant, ranging from 279 to 282 bp. Multiple copies of divergent ITS1 present in the tomato genome indicate that some copies may be paralogues. In conclusion, DGGE technique is a reliable and novel approach to reveal the entire ITS copy variation and the possible evolutionary relationship of tomato.