一种提高光呼吸改良植物耐旱性的方法
阅读说明:本技术 一种提高光呼吸改良植物耐旱性的方法 (Method for improving drought tolerance of plants through light respiration ) 是由 王东芳 张先文 沈志成 于 2019-08-13 设计创作,主要内容包括:本发明公开了一种提高光呼吸改良植物耐旱性的方法,所述方法为:抑制或敲除植物中胆汁酸钠协同转运蛋白基因,同时过表达乙醛酸脱氢酶基因和苹果酸合酶基因。本发明创造性的发现抑制植物中BASS6基因的表达,结合在叶绿体中过表达乙醇酸脱氢酶基因、苹果酸合酶基因,能显著减少光呼吸,提高植物的光合效率,提高植物生物量或产量,更重要的是这种转基因植物的耐旱性显著高于抑制PLGG1基因表达的植株,耐旱性提高5%-50%。(The invention discloses a method for improving drought tolerance of plants improved by light respiration, which comprises the following steps: inhibiting or knocking out sodium bile cotransporter gene in plant, and over-expressing glyoxylate dehydrogenase gene and malic acid synthase gene. The invention creatively discovers that the expression of BASS6 gene in the plant is inhibited, and the overexpression of glycollic dehydrogenase gene and malic acid synthase gene in chloroplast can obviously reduce the light respiration, improve the photosynthetic efficiency of the plant and improve the biomass or yield of the plant, more importantly, the drought tolerance of the transgenic plant is obviously higher than that of the plant inhibiting the expression of PLGG1 gene, and the drought tolerance is improved by 5-50%.)
(一)技术领域
本发明涉及一种提高光呼吸改良植物抗旱性的方法,通过转基因的方法来提高植物的光合效率、生物量或产量,改良植物种植资源。
(二)背景技术
人类数量的增加和生活水平的提高,需要消耗更多的粮食和饲料,这就要求在有限的土地上收获更多粮食。因此,培育新的高产植物品种非常重要。
植物整体的光合作用产物全部来源于酶催化CO2转化为有机碳化合物。1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)是卡尔文循环(Calvin-Benson(CB)cycle)中的羧化酶。由于RubisCO与CO2或O2都能反应,RubisCO与O2反应产生磷酸乙醇酸,进入光呼吸循环,光呼吸导致植物中固定的碳和氮的浪费。在全球范围内,这一过程每年将大约29 GT的新鲜同化碳被重新释放到大气中(Anav A,etal.Spatiotemporal patterns of terrestrial grossprimary production:a review.Rev Geophys 2015,
53:785-818.)。
为了减少光呼吸造成的损失,提高植物的光合效率,目前常用的方法是通过新的光呼吸支路来回收乙醇酸中的CO2,从而达到减少光呼吸提高光合效率的目的(Peterhansel C,Blume C,Offermann S.Photorespiratory bypasses:how can theywork?[J].Journal of Experimental Botany,2013,64(3):709-715.)。
乙醇酸脱氢酶(glycolate dehydrogenase,GDH)可以将乙醇酸转换成乙醛酸。目前用于植物转基因研究和应用的乙醇酸脱氢酶主要是来源于低等植物绿藻(Chlamydomonasreinhardtii)或者大肠杆菌。绿藻中的乙醇酸脱氢酶由一个基因编码,而大杆菌中的乙醇酸脱氢酶分别由3个基因编码的D、E、F三个亚基构成。有报到通过在土豆中过表达D、E、F三个亚基的编码基因的融合基因后,植株中的DEFp融合蛋白表达量增加,葡萄糖、果糖和蔗糖等糖分成倍增加,生物量也显著增加(Nolke G,Houdelet M,Kreuzaler F,et al.The expression of a recombinant glycolate dehydrogenase polyprotein inpotato(Solanumtuberosum)plastids strongly enhances photosynthesis and tuberyield[J].Plant Biotechnology Journal,2014,12(6):734-742.)。但是大肠杆菌来源和绿藻来源的乙醇酸脱氢酶在功能和活性方面都有显著差异,所以在转基因植物中的表现也有很大的差异。
苹果酸合酶(malate synthase,MS)能催化乙酰-CoA与乙醛酸转换成苹果酸与CoA。苹果酸合酶参与了乙醛酸循环,广泛存在于不同植物体内。有研究报道在烟草中过表达绿藻的GDH和南瓜(C.maxima)的MS可以提高其光合效率和生物量(PF South,APCavanagh,HW Liu,etal.Synthetic glycolate metabolism pathways stimulate cropgrowth and productivity in the field,Science,2019:363(6422):eaat9077.)。
胆汁酸钠协同转运蛋白(Bile Acid Sodium Symporter,BASS)和质体乙醇酸/甘油酸转运子1(plastidalglycolate/glyceratetranslocator 1,PLGG1)是光呼吸中,把叶绿体中的乙醇酸转运到过氧化物酶体的关键蛋白质(South P F,Walker B J,Cavanagh AP,et al.Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and IsInvolved in Photorespiratory Metabolism in Arabidopsis thaliana[J].The PlantCell,2017:tpc.00775.2016.)。BASS和PLGG1基因都有转运乙醇酸的功能,但是PLGG1同时还具有转运乙醇酸和甘油酸的功能。之前的研究表明在烟草叶绿体中过表达GDH和MS基因,同时抑制PLGG1基因的表达,可以显著提高烟草的生物量(PF South,AP Cavanagh,HW Liu,etal.Synthetic glycolate metabolism pathways stimulate crop growth andproductivity in the field,Science,2019:363(6422):eaat9077.)。
耐旱是植物非常重要的特性。植物具有一定的耐旱性有利于其抵抗干旱逆境,适应不同地理环境。在水资源日益紧张的情况下,培育耐旱能力强的作物新品种非常重要。
但是我们研究发现,在叶绿体中过表达GDH和MS基因,同时抑制PLGG1基因的表达的植物与对照相比,耐旱性显著降低。相比之下,在叶绿体中过表达GDH和MS基因,同时抑制BASS6基因的表达的植物的耐旱性显著高于抑制PLGG1基因的表达的植物。
(三)
发明内容
本发明目的是提供一种保持或提高植物耐旱性,且减少植物光呼吸,提高植物光合效率,提高植物生物量或产量的方法,为了实现上述技术目的,本发明的技术手段是抑制或敲除植物中BASS基因的表达,并在叶绿体中过表达GDH、MS基因。
本发明采用的技术方案是:
本发明提供一种提高光呼吸改良植物耐旱性的方法,所述方法为:抑制或敲除植物中胆汁酸钠协同转运蛋白(BASS)基因,同时过表达乙醛酸脱氢酶(GDH)基因和苹果酸合酶(MS)基因。
进一步,所述胆汁酸钠协同转运蛋白的编码基因来源于植物(表1),其氨基酸序列如SEQ ID NO.1,SEQ ID NO.2或SEQ ID NO.3之一所示。当所述植物是水稻时,其BASS基因的氨基酸序列为SEQ ID NO.1,当所述植物是大豆时,其BASS基因的氨基酸序列为SEQ IDNO.2或3。
进一步,抑制植物中胆汁酸钠协同转运蛋白基因的方法为RNA干扰法,具体是向植物中导入形成靶向胆汁酸钠协同转运蛋白基因发夹结构的双链RNA核苷酸序列。优选靶向水稻OsBASS基因和大豆GmBASS基因形成发夹结构的OsBASS-RNAi和GmBASS-RNAi序列,序列如SEQ ID NO.4和SEQ ID NO.5所示。
进一步,所述乙醇酸脱氢酶GDH基因(表2)可以来源于原核生物或者真核生物,包括但不限于表2中所示的基因,核苷酸序列如SEQ ID NO.6(氨基酸序列为SEQ ID NO.7)。
进一步,所述苹果酸合酶(MS)(表3)可以来源于原核生物或者真核生物,包括但不限于表3中所示的基因,优选MS的核苷酸序列如SEQ ID NO.8所示,其氨基酸序列如SEQ IDNO.9所示。
本发明所述方法是构建T-DNA载体,导入植物中完成;所述T-DNA载体构建方法为:以含有耐草铵膦bar基因的pCambia1300双元载体为基础载体,再分别连入乙醇酸脱氢酶基因表达框、苹果酸合酶基因表达框和胆汁酸钠协同转运蛋白基因RNAi表达框;所述含有耐草铵膦bar基因的pCambia1300双元载体是将抗潮霉素基因hptII替换为抗草铵膦基因bar。
本发明中BASS基因RNAi表达框,GDH基因过表达框、MS基因过表达框可以通过分子聚合或者杂交聚合的方法实现。分子聚合是指将BASS基因RNAi表达框、GDH、MS基因的表达框构建在同一个载体的T-DNA上,通过转基因的方法将T-DNA转入的受体植物基因组中,从而使得目标植株中同时对BASS基因的表达进行抑制,且过表达GDH基因、MS基因。杂交聚合是指分别获得包含BASS基因抑制表达框、GDH过表达框、MS过表达框的植株,再用传统育种的方法,将分别含有一个或两个上述表达框的植株进行杂交,获得同时含有上述3个表达框的转基因植株。
本发明介导GDH、MS基因在叶绿体中过表达的信号肽来源于植物RuBisCO小亚基(RbcS)或磷酸葡萄糖变位酶转运肽序列,优选叶绿体信号肽的氨基酸序列如SEQ ID NO.10或SEQ ID NO.11所示,叶绿体信号肽序列融合在GDH或MS蛋白的N端。本发明介导GDH、MS过表达的启动子可以来源于真核生物或者原核生物,也可以通过人工合成获得,可以是组成型启动子或者特异性启动子。所述的启动子包括p35S(NCBI ACCESSION:MG719235 REGION:848-1628),玉米UBI启动子(NCBI ACCESSION:KR297238 REGION:4879-6876)和水稻Actin1启动子(NCBI ACCESSION:AY452735 REGION:2428-3797)。
本发明本发明介导GDH、MS过表达的终止子可以来源于真核生物或者原核生物,也可以通过人工合成获得,优选终止子为ter1(NCBI ACCESSION:KJ716235REGION:3962-4158)和ter2(NCBI ACCESSION:MG733984 REGION:2092-2314)。
本发明所述植物为碳三植物,是指CO2同化的最初产物是光合碳循环中的三碳化合物3-磷酸甘油酸的植物,主要包括水稻和大豆。
本发明提供了一种提高植物耐旱性的方法,通过对植物BASS基因进行RNA干扰,并结合在植物的叶绿体中过表达乙醛酸脱氢酶基因(GDH)和苹果酸合酶基因(MS),在叶绿体中将乙醇酸转化为乙醛酸并进一步转化为苹果酸,苹果酸在植物叶绿体自身的苹果酸酶的作用下变成丙酮酸,再在丙酮酸脱氢酶的作用下转变成乙酰辅酶A,从而达到减少光呼吸、提高光合效率和产量的目的。在水稻和大豆叶绿体中对BASS基因进行RNA干扰,并过表达乙醛酸脱氢酶基因和苹果酸合酶,使得水稻产量增加3%-50%,大豆产量增加3%-50%,且耐旱性与非转基因对照相当,或者比对照更好。
表1:胆汁酸钠协同转运蛋白(BASS)基因
编号
来源物种
NCBI Accession Number
1
Arabidopsisthaliana
<u>NP</u> 567671
2
Zeamays
<u>NP</u> 001158917
3
Sorghumbicolor
XP 021308938
4
Oryzasativa
<u>XP</u>015612294
5
Glycinemax
XP 003538535/XP 003517442
表2:不同物种来源的乙醇酸脱氢酶(GDH)基因
编号
来源物种
NCBI Accession Number
1
Chlamydomonas reinhardtii
<u>XP 001695381.1</u>
2
Volvox carteri f.nagariensis
<u>XP002946459.1</u>
3
Gonium pectorale
<u>KXZ46746.1</u>
4
Chlamydomonas eustigma
<u>GAX77289.1</u>
5
Escherichia coli K-12
<u>NP</u> 417453.1、YP 026191.1、YP 026190.1
表3:不同物种来源的苹果酸合酶(MS)基因
与现有技术相比,本发明有益效果主要体现在:
本发明创造性的发现抑制植物中BASS6基因的表达,结合在叶绿体中过表达乙醇酸脱氢酶基因(GDH)、苹果酸合酶基因(MS),能显著减少光呼吸,提高植物的光合效率,提高植物生物量或产量,更重要的是这种转基因植物的耐旱性显著高于抑制PLGG1基因表达的植株,耐旱性提高3%-50%。
(四)
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1、载体的构建
GDH基因可以来源原核生物,也可以来源于真核生物,本发明提供的GDH基因包括但不限于表2中所示的基因。为了构建转化载体,人工合成了大肠杆菌来源的GDH基因以及对应的终止子序列,包含叶绿体信号肽、GDH编码基因和终止子,核苷酸序列如SEQ ID NO.6所示(氨基酸序列为SEQ ID NO.7所示),5‘端和3‘端分别设置有BamHI和KpnI位点。
MS基因可以来源原核生物,也可以来源于真核生物,本发明提供的MS基因包括但不限于表3中所示的基因。人工合成了水稻的MS基因,包含叶绿体信号肽、MS编码基因和终止子,核苷酸序列如SEQ ID NO.8所示(氨基酸序列为SEQ ID NO.9所示),5‘端和3‘端分别设置有BamHI和HindIII位点。
为了实现对BASS基因的表达抑制或敲除,本发明提供了植物自身的BASS基因(表1),当所述植物是水稻时,其BASS基因的氨基酸序列为SEQ ID NO.1,当所述植物是大豆时,其BASS基因的氨基酸序列为SEQ ID NO.2或3。为了构建BASS基因干扰表达框,分别人工合成靶向水稻OsBASS基因和大豆GmBASS基因的可以形成发夹结构的OsBASS-RNAi和GmBASS-RNAi序列,序列如SEQ ID NO.4和SEQ ID NO.5所示。作为对照分别合成了靶向水稻OsPGGL1基因和大豆GmPGGL1基因的可以形成发夹结构的Os PGGL1-RNAi和Gm PGGL1-RNAi序列,如SEQ ID NO.12和SEQ ID NO.13所示。上述序列的3’端都分别加有终止子ter1,最终构成OsBASS-RNAi-ter、GmBASS-RNAi-ter和Os PGGL1-RNAi-ter、Gm PGGL1-RNAi-ter。5‘端和3‘端分别设置有BglII和HindIII位点。
同时,人工合成花椰菜花叶病毒(CaMV)的35S和玉米Ubi启动子序列。35S启动子5‘端和3‘端分别设置有EcoRI和BamHI位点,Ubi启动子5‘端和3‘端分别设置有HindIII和BamHI位点,用于介导RNAi序列的转录。
为了构建可以用于农杆菌方法转化植物所用的双元载体,用商业化的双元载体pCambia1300为基础,通过XhoI酶切位点把之前的hptII(hygromycin resistance)基因置换成耐草铵膦的bar基因(NCBI ACCESSIONp:MG719235 REGION:287-835),置换后的载体命名为pCambia1300-bar。
通过EcoRI和KpnI位点把Ubi启动子与SEQ ID NO.6所示GDH基因连入pCambia1300-bar载体中,获得过度载体pCambia1300-bar-GDH。再通过KpnI和HindIII位点把35S启动子和SEQ ID NO.10所示MS基因连入过度载体pCambia1300-bar-GDH中,获得过度载体pCambia1300-bar-GDH-MS。最后通过HindIII对pCambia1300-bar-GDH-MS进行单酶切,再把用BglII和HindIII双酶切后的OsBASS-RNAi-ter或GmBASS-RNAi-ter和用BamHI和HindIII酶切后的Ubi启动子连接,构建成终载体,分别命名为pCambia1300-bar-GDH-MS-OsBASS-RNAi(GMOsBi)和pCambia1300-bar-GDH-MS-GmBASS-RNAi(GMGmBi)。
作为对照,用同样的方法构建含有抑制PGGL1基因表达框的载体,分别命名为pCambia1300-bar-GDH-MS-OsPGGL1-RNAi(GMOsPi)和pCambia1300-bar-GDH-MS-GmPGGL1-RNAi(GMGmPi)。
最后,通过电转的方法把T-DNA质粒转入农杆菌LB4404中,通过含有15μg/ml四环素和50μg/mL的卡那霉素的YEP固体培养基筛选出阳性克隆,并保菌,用于接下来的植物转化。
实施例2、水稻转化
转基因水稻的获得方法是采用现有技术(卢雄斌,龚祖埙(1998)生命科学10:125-131;刘凡等(2003)分子植物育种1:108-115)。选取成熟饱满的“秀水-134”种子去壳,诱导产生愈伤组织作为转化材料。取实施例1中构建好的分别含有pCambia1300-bar-GDH-MS-OsBASS-RNAi(GMOsBi)和pCambia1300-bar-GDH-MS-OsPGGL1-RNAi(GMOsPi)质粒的农杆菌划板。挑单菌落接种,准备转化用农杆菌。将待转化的愈伤组织放入OD为0.6左右的农杆菌菌液中(农杆菌菌液的制备:将农杆菌接种至培养基,培养至OD为0.6左右;培养基组成:3g/L K2HPO4、1g/L NaH2PO4、1g/L NH4Cl、0.3g/L MgSO4·7H2O、0.15g/L KCl、0.01g/L CaCl2、0.0025g/L FeSO4·7H2O、5g/L蔗糖、20mg/L乙酰丁香酮,溶剂为水,pH=5.8),让农杆菌结合到愈伤组织表面,然后把愈伤组织转移到共培养培养基(MS+2mg/L 2,4-D(2,4-二氯苯氧乙酸)+30g/L葡萄糖+30g/L蔗糖+3g/L琼脂(sigma 7921)+20mg/L乙酰丁香酮)中,共培养2-3天。用无菌水冲洗转化后的愈伤,转移到筛选培养基(MS+2mg/L 2,4-D+30g/L蔗糖+3g/L琼脂(sigma 7921)+20mg/L乙酰丁香酮+2mM草甘膦(Sigma))上,筛选培养两个月(中间继代一次)。把筛选后,生长活力良好的愈伤转移到预分化培养基(MS+0.1g/L肌醇+5mg/L ABA(脱落酸)+1mg/L NAA(萘乙酸)+5mg/L 6-BA(6-苄胺基腺嘌呤)+20g/L山梨醇+30g/L蔗糖+2.5g/L植物凝胶(gelrite))上培养20天左右,然后将预分化好的愈伤组织移到分化培养基上,每天14小时光照分化发芽。2-3周后,把抗性再生植株转移到生根培养基(1/2 MS+0.2mg/L NAA+20g/L蔗糖+2.5g/L gelrite)上壮苗生根,最后将再生植株洗去琼脂移植于温室,选择产量高、种子大或者生物量高等能够提高水稻产量的转基因株系,培育新品种。获得含上述转化载体的转基因水稻植株。
实施例3.大豆转化
这里使用的获得转基因大豆的步骤来自于已有的技术(Deng et al.,1998,PlantPhysiology Communications 34:381-387;Ma et al.,2008,ScientiaAgriculturaSinica 41:661-668;Zhou et al.,2001,Journal of NortheastAgricultural University 32:313-319)。选取健康、饱满、成熟的“天隆1号”大豆,用80%乙醇消毒2分钟,再用无菌水清洗,然后放置在充满氯气(由50ml NaClO与2ml浓HCl反应生成)的干燥器中灭菌4-6个小时。灭菌后的大豆在超净工作台里被播撒到B5培养基中,25℃条件下培养5天,同时光密度在90-150μmol光子/m2·s水平。当子叶变绿并顶破种皮,无菌的豆芽就会长出。去掉了下胚轴的豆芽在长度上被切成五五开,使得两片外植体都具有子叶和上胚轴。在子叶和上胚轴的节点处切外植体大约7-8处,即可用作被侵染的目标组织。
分别取通过实施例1构建的含有载体pCambia1300-bar-GDH-MS-GmBASS-RNAi(GMGmBi)和pCambia1300-bar-GDH-MS-GmPGGL1-RNAi(GMGmPi)的单克隆农杆菌被分开培养待用。准备好的外植体浸没在农杆菌悬浮液中共培养30分钟左右。然后,将侵染的组织上多余的细胞悬浮液用吸水纸吸收干净,再转移到1/10 B5共培养培养基里25℃暗培养3-5天。
共培养的植物组织用B5液体培养基清洗,以除去多余的农杆菌,然后放置到B5固体培养基中25℃下培养5天,待其发芽。诱导发生的胚芽组织转移到含有0.2mM草甘膦的B5筛选培养基中,25℃光照培养4周,期间每两周更换一次培养基。筛选出来的胚芽组织再转移到固体培养基中,25℃培养,待其长成小苗。随后,将转基因植株苗转移到1/2 B5培养基中进行生根诱导。最后,长成的小植株经清洗去除琼脂后栽种在温室中。
实施例4:转基因水稻的鉴定
通过实施例2分别获得了载体pCambia1300-bar-GDH-MS-OsBASS-RNAi(GMOsBi)和pCambia1300-bar-GDH-MS-OsPGGL1-RNAi(GMOsPi)的转基因水稻植株。上述转基因植株与非转基因对照相比生物量和产量都有所增加,并且GMOsBi植株在生物量或产量方面的增加幅度最大。为了进一步鉴定GMOsBi转基因植株的表现变化,对上述转基因植株的生物量和种子产量进行了评估和比较,根据表6农业农村部制定的《基于农田与作物干旱形态指标等级》,设置了正常水分条件、轻旱条件和中旱条件,结果如表4所示。在正常条件下GMOsBi转基因植株和GMOsPi转基因植株的生物量或产量与非转基因对照相比都有显著增加,增幅达5%-50%;但是在轻旱条件下,GMOsBi转基因植株与对照相比生物量或产量仍然显著增加,而GMOsPi转基因植株与非转基因对照相比生物量或产量增加非常少;而在中旱条件下,GMOsBi转基因植株与对照相比生物量或产量仍然显著增加,而GMOsPi转基因植株与非转基因对照相比生物量或产量没有任何增加,甚至有所降低。上述结果表明,在干旱条件下,本发明的GMOsBi转基因植株的抗旱性显著优于对照方案的转基因植株。
表4转基因水稻在不同干旱条件下的产量测定
实施例5:转基因大豆的鉴定
通过实施例3分别获得了载体pCambia1300-bar-GDH-MS-GmBASS-RNAi(GMGmBi)和pCambia1300-bar-GDH-MS-GmPGGL1-RNAi(GMGmPi)的转基因大豆植株。上述转基因植株与非转基因对照相比生物量和产量都有所增加,并且,转基因植株在生物量或产量方面的增加幅度最大。为了进一步鉴定转基因植株的表现变化,对上述转基因植株的生物量和种子产量进行了评估,根据表6农业农村部制定的《基于农田与作物干旱形态指标等级》,设置了正常水分条件、轻旱条件和中旱条件,结果如表5所示。在正常条件下GMGmBi转基因植株和GMGmPi转基因植株的生物量或产量与非转基因对照相比都有显著增加,增幅达5%-50%;但是在轻旱条件下,GMGmBi转基因植株与对照相比生物量或产量仍然显著增加,而GMGmPi转基因植株与非转基因对照相比生物量或产量增加非常少;而在中旱条件下,GMGmBi转基因植株与对照相比生物量或产量仍然显著增加,而GMGmPi转基因植株与非转基因对照相比生物量或产量没有任何增加,甚至有所降低。上述结果表明在干旱条件下,本发明的GMGmBi转基因植株的抗旱性显著优于对照方案的转基因植株。
表5转基因大豆在不同干旱条件下的产量测定
表6、基于农田与作物干旱形态指标等级(GB/T 32136-2015)
序列表
<110> 浙江大学
<120> 一种提高光呼吸改良植物耐旱性的方法
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ccaccagaag aagaacaagc tcccgatcca gtacaagatc ggcccggacc cgtcctccat 900
cgactcctgc atgatcggcg gcatcgtgtc caacaactcc tccggcatgt gctgcggcgt 960
gtcccagaac acctaccaca ccctcaagga catgcgcgtg gtgttcgtgg acggcaccgt 1020
gctcgacacc gccgacccga actcctgcac cgccttcatg aagtcccacc gctccctcgt 1080
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ccgccgcaag ttcgccatca agtgcaccac cggctactcc ctcaacgccc tcgtggactt 1200
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cggcttcgtg tcccgcgcca cctacaacac cgtgccggag tggccgaaca aggcctccgc 1320
cttcatcgtg ttcccggacg tgcgcgccgc ctgcaccggc gcctccgtgc tccgcaacga 1380
gacctccgtg gacgccgtgg agctcttcga ccgcgcctcc ctccgcgagt gcgagaacaa 1440
cgaggacatg atgcgcctcg tgccggacat caagggctgc gacccgatgg ccgccgccct 1500
cctcatcgag tgccgcggcc aggacgaggc cgccctccag tcccgcatcg aggaggtggt 1560
gcgcgtgctc accgccgccg gcctcccgtt cggcgccaag gccgcccagc cgatggccat 1620
cgacgcctac ccgttccacc acgaccagaa gaacgccaag gtgttctggg acgtgcgccg 1680
cggcctcatc ccgatcgtgg gcgccgcccg cgagccgggc acctccatgc tcatcgagga 1740
cgtggcctgc ccggtggaca agctcgccga catgatgatc gacctcatcg acatgttcca 1800
gcgccacggc taccacgacg cctcctgctt cggccacgcc ctcgagggca acctccacct 1860
cgtgttctcc cagggcttcc gcaacaagga ggaggtgcag cgcttctccg acatgatgga 1920
ggagatgtgc cacctcgtgg ccaccaagca ctccggctcc ctcaagggcg agcacggcac 1980
cggccgcaac gtggccccgt tcgtggagat ggagtggggc aacaaggcct acgagctcat 2040
gtgggagctc aaggccctct tcgacccgtc ccacaccctc aacccgggcg tgatcctcaa 2100
ccgcgaccag gacgcccaca tcaagttcct caagccgtcc ccggccgcct ccccgatcgt 2160
gaaccgctgc atcgagtgcg gcttctgcga gtccaactgc ccgtcccgcg acatcaccct 2220
caccccgcgc cagcgcatct ccgtgtaccg cgagatgtac cgcctcaagc agctcggccc 2280
gggcgcctcc gaggaggaga agaagcagct cgccgccatg tcctcctcct acgcctacga 2340
cggcgagcag acctgcgccg ccgacggcat gtgccaggag aagtgcccgg tgaagatcaa 2400
caccggcgac ctcatcaagt ccatgcgcgc cgagcacatg aaggaggaga agaccgcctc 2460
cggcatggcc gactggctcg ccgccaactt cggcgtgatc aactccaacg tgccgcgctt 2520
cctcaacatc gtgaacgcca tgcactccgt ggtgggctcc gccccgctct ccgccatctc 2580
ccgcgccctc aacgccgcca ccaaccactt cgtgccggtg tggaacccgt acatgccgaa 2640
gggcgccgcc ccgctcaagg tgccggcccc gccggccccg gccgccgccg aggcctccgg 2700
catcccgcgc aaggtggtgt acatgccgtc ctgcgtgacc cgcatgatgg gcccggccgc 2760
ctccgacacc gagaccgccg ccgtgcacga gaaggtgatg tccctcttcg gcaaggccgg 2820
ctacgaggtg atcatcccgg agggcgtggc ctcccagtgc tgcggcatga tgttcaactc 2880
ccgcggcttc aaggacgccg ccgcctccaa gggcgccgag ctcgaggccg ccctcctcaa 2940
ggcctccgac aacggcaaga tcccgatcgt gatcgacacc tccccgtgcc tcgcccaggt 3000
gaagtcccaa atctccgagc cgtccctccg cttcgccctc tacgagccgg tggagttcat 3060
ccgccacttc ctcgtggaca agctcgagtg gaagaaggtg cgcgaccagg tggccatcca 3120
cgtgccgtgc tcctccaaga agatgggcat cgaggagtcc ttcgccaagc tcgccggcct 3180
ctgcgccaac gaggtggtgc cgtccggcat cccgtgctgc ggcatggccg gcgaccgcgg 3240
catgcgcttc ccggagctca ccggcgcctc cctccagcac ctcaacctcc cgaagacctg 3300
caaggacggc tactccacct cccgcacctg cgagatgtcc ctctccaacc acgccggcat 3360
caacttccgc ggcctcgtgt acctcgtgga cgaggccacc gccccgaaga agcaggccgc 3420
cgccgccaag accgcctaag tagatgccga ccggatctgt cgatcgacaa gctcgagttt 3480
ctccataata atgtgtgagt agttcccaga taagggaatt agggttccta tagggtttcg 3540
ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta 3600
tcaataaaat ttctaattcc taaaaccaaa atccagtact aaaatccaga tcccccgaat 3660
taaagctt 3668
<210> 7
<211> 1136
<212> PRT
<213> 未知(Unknown)
<400> 7
Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala
1 5 10 15
Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala
20 25 30
Phe Pro Ala Thr Arg Lys Ala Asn Gly Gly Pro Arg Gly Gln Gly Lys
35 40 45
Arg Leu Ala Gln Leu Leu Gly Ala Gln Leu Lys Gln Tyr Ala Ala Glu
50 55 60
Val Arg Gly Ile Ser Thr Ala Gly Gly Ala Ser Arg Gly Gly Ala Arg
65 70 75 80
Gly Pro Ala Ser Pro Ser Ser Leu Glu Gln Gln Thr Arg Gln Val Ala
85 90 95
Gln Val Ala Val Gln Gln Ser Thr Gln Gln Ala Val Lys Val Val Val
100 105 110
Pro Ala Ile Lys Val Asp Leu Val Gly Ala Val Ser Ser Val Ser Glu
115 120 125
Ser Asp Lys Val Glu Pro Gly Val Phe Lys Asn Val Asp Gly His Arg
130 135 140
Phe Glu Asp Gly Arg Tyr Ala Ala Phe Val Glu Glu Ile Thr Lys Phe
145 150 155 160
Ile Pro Lys Glu Arg Gln Tyr Ser Asp Pro Val Arg Thr Phe Ala Tyr
165 170 175
Gly Thr Asp Ala Ser Phe Tyr Arg Leu Asn Pro Lys Leu Val Val Lys
180 185 190
Val His Asn Glu Asp Glu Val Arg Arg Ile Met Pro Ile Ala Glu Arg
195 200 205
Leu Gln Val Pro Ile Thr Phe Arg Ala Ala Gly Thr Ser Leu Ser Gly
210 215 220
Gln Ala Ile Thr Asp Ser Val Leu Ile Lys Leu Ser His Thr Gly Lys
225 230 235 240
Asn Phe Arg Asn Phe Thr Val His Gly Asp Gly Ser Val Ile Thr Val
245 250 255
Glu Pro Gly Leu Ile Gly Gly Glu Val Asn Arg Ile Leu Ala Ala His
260 265 270
Gln Lys Lys Asn Lys Leu Pro Ile Gln Tyr Lys Ile Gly Pro Asp Pro
275 280 285
Ser Ser Ile Asp Ser Cys Met Ile Gly Gly Ile Val Ser Asn Asn Ser
290 295 300
Ser Gly Met Cys Cys Gly Val Ser Gln Asn Thr Tyr His Thr Leu Lys
305 310 315 320
Asp Met Arg Val Val Phe Val Asp Gly Thr Val Leu Asp Thr Ala Asp
325 330 335
Pro Asn Ser Cys Thr Ala Phe Met Lys Ser His Arg Ser Leu Val Asp
340 345 350
Gly Val Val Ser Leu Ala Arg Arg Val Gln Ala Asp Lys Glu Leu Thr
355 360 365
Ala Leu Ile Arg Arg Lys Phe Ala Ile Lys Cys Thr Thr Gly Tyr Ser
370 375 380
Leu Asn Ala Leu Val Asp Phe Pro Val Asp Asn Pro Ile Glu Ile Ile
385 390 395 400
Lys His Leu Ile Ile Gly Ser Glu Gly Thr Leu Gly Phe Val Ser Arg
405 410 415
Ala Thr Tyr Asn Thr Val Pro Glu Trp Pro Asn Lys Ala Ser Ala Phe
420 425 430
Ile Val Phe Pro Asp Val Arg Ala Ala Cys Thr Gly Ala Ser Val Leu
435 440 445
Arg Asn Glu Thr Ser Val Asp Ala Val Glu Leu Phe Asp Arg Ala Ser
450 455 460
Leu Arg Glu Cys Glu Asn Asn Glu Asp Met Met Arg Leu Val Pro Asp
465 470 475 480
Ile Lys Gly Cys Asp Pro Met Ala Ala Ala Leu Leu Ile Glu Cys Arg
485 490 495
Gly Gln Asp Glu Ala Ala Leu Gln Ser Arg Ile Glu Glu Val Val Arg
500 505 510
Val Leu Thr Ala Ala Gly Leu Pro Phe Gly Ala Lys Ala Ala Gln Pro
515 520 525
Met Ala Ile Asp Ala Tyr Pro Phe His His Asp Gln Lys Asn Ala Lys
530 535 540
Val Phe Trp Asp Val Arg Arg Gly Leu Ile Pro Ile Val Gly Ala Ala
545 550 555 560
Arg Glu Pro Gly Thr Ser Met Leu Ile Glu Asp Val Ala Cys Pro Val
565 570 575
Asp Lys Leu Ala Asp Met Met Ile Asp Leu Ile Asp Met Phe Gln Arg
580 585 590
His Gly Tyr His Asp Ala Ser Cys Phe Gly His Ala Leu Glu Gly Asn
595 600 605
Leu His Leu Val Phe Ser Gln Gly Phe Arg Asn Lys Glu Glu Val Gln
610 615 620
Arg Phe Ser Asp Met Met Glu Glu Met Cys His Leu Val Ala Thr Lys
625 630 635 640
His Ser Gly Ser Leu Lys Gly Glu His Gly Thr Gly Arg Asn Val Ala
645 650 655
Pro Phe Val Glu Met Glu Trp Gly Asn Lys Ala Tyr Glu Leu Met Trp
660 665 670
Glu Leu Lys Ala Leu Phe Asp Pro Ser His Thr Leu Asn Pro Gly Val
675 680 685
Ile Leu Asn Arg Asp Gln Asp Ala His Ile Lys Phe Leu Lys Pro Ser
690 695 700
Pro Ala Ala Ser Pro Ile Val Asn Arg Cys Ile Glu Cys Gly Phe Cys
705 710 715 720
Glu Ser Asn Cys Pro Ser Arg Asp Ile Thr Leu Thr Pro Arg Gln Arg
725 730 735
Ile Ser Val Tyr Arg Glu Met Tyr Arg Leu Lys Gln Leu Gly Pro Gly
740 745 750
Ala Ser Glu Glu Glu Lys Lys Gln Leu Ala Ala Met Ser Ser Ser Tyr
755 760 765
Ala Tyr Asp Gly Glu Gln Thr Cys Ala Ala Asp Gly Met Cys Gln Glu
770 775 780
Lys Cys Pro Val Lys Ile Asn Thr Gly Asp Leu Ile Lys Ser Met Arg
785 790 795 800
Ala Glu His Met Lys Glu Glu Lys Thr Ala Ser Gly Met Ala Asp Trp
805 810 815
Leu Ala Ala Asn Phe Gly Val Ile Asn Ser Asn Val Pro Arg Phe Leu
820 825 830
Asn Ile Val Asn Ala Met His Ser Val Val Gly Ser Ala Pro Leu Ser
835 840 845
Ala Ile Ser Arg Ala Leu Asn Ala Ala Thr Asn His Phe Val Pro Val
850 855 860
Trp Asn Pro Tyr Met Pro Lys Gly Ala Ala Pro Leu Lys Val Pro Ala
865 870 875 880
Pro Pro Ala Pro Ala Ala Ala Glu Ala Ser Gly Ile Pro Arg Lys Val
885 890 895
Val Tyr Met Pro Ser Cys Val Thr Arg Met Met Gly Pro Ala Ala Ser
900 905 910
Asp Thr Glu Thr Ala Ala Val His Glu Lys Val Met Ser Leu Phe Gly
915 920 925
Lys Ala Gly Tyr Glu Val Ile Ile Pro Glu Gly Val Ala Ser Gln Cys
930 935 940
Cys Gly Met Met Phe Asn Ser Arg Gly Phe Lys Asp Ala Ala Ala Ser
945 950 955 960
Lys Gly Ala Glu Leu Glu Ala Ala Leu Leu Lys Ala Ser Asp Asn Gly
965 970 975
Lys Ile Pro Ile Val Ile Asp Thr Ser Pro Cys Leu Ala Gln Val Lys
980 985 990
Ser Gln Ile Ser Glu Pro Ser Leu Arg Phe Ala Leu Tyr Glu Pro Val
995 1000 1005
Glu Phe Ile Arg His Phe Leu Val Asp Lys Leu Glu Trp Lys Lys Val
1010 1015 1020
Arg Asp Gln Val Ala Ile His Val Pro Cys Ser Ser Lys Lys Met Gly
1025 1030 1035 1040
Ile Glu Glu Ser Phe Ala Lys Leu Ala Gly Leu Cys Ala Asn Glu Val
1045 1050 1055
Val Pro Ser Gly Ile Pro Cys Cys Gly Met Ala Gly Asp Arg Gly Met
1060 1065 1070
Arg Phe Pro Glu Leu Thr Gly Ala Ser Leu Gln His Leu Asn Leu Pro
1075 1080 1085
Lys Thr Cys Lys Asp Gly Tyr Ser Thr Ser Arg Thr Cys Glu Met Ser
1090 1095 1100
Leu Ser Asn His Ala Gly Ile Asn Phe Arg Gly Leu Val Tyr Leu Val
1105 1110 1115 1120
Asp Glu Ala Thr Ala Pro Lys Lys Gln Ala Ala Ala Ala Lys Thr Ala
1125 1130 1135
<210> 8
<211> 2057
<212> DNA
<213> 未知(Unknown)
<400> 8
ggatccaaca atggccccgt ccgtgatggc ctcctccgcc accaccgtgg ccccgttcca 60
gggcctcaag tccaccgccg gcatgccggt ggcccgccgc tccggcaact cctccttcgg 120
caacgtgtcc aacggcggcc gcatccgctg catggccacc aacgccgccg ccccgccgtg 180
cccgtgctac gacaccccgg agggcgtgga catcctcggc cgctacgacc cggagttcgc 240
cgccatcctc acccgcgacg ccctcgcctt cgtggccggc ctccagcgcg agttccgcgg 300
cgccgtgcgc tacgccatgg agcgccgccg cgaggcccag cgccgctacg acgccggcga 360
gctcccgcgc ttcgacccgg ccacccgccc ggtgcgcgag gccggcggct gggcctgcgc 420
cccggtgccg ccggccatcg ccgaccgcac cgtggagatc accggcccgg ccgagccgcg 480
caagatggtg atcaacgccc tcaactccgg cgccaaggtg ttcatggccg acttcgagga 540
cgccctctcc ccgacctggg agaacctcat gcgcggccag gtgaacctcc gcgacgccgt 600
ggccggcacc atcacctacc gcgacgccgc ccgcggccgc gagtaccgcc tcggcgaccg 660
cccggccacc ctcttcgtgc gcccgcgcgg ctggcacctc ccggaggccc acgtgctcgt 720
ggacggcgag ccggccatcg gctgcctcgt ggacttcggc ctctacttct tccactccca 780
cgccgccttc cgctccggcc agggcgccgg cttcggcccg ttcttctacc tcccgaagat 840
ggagcactcc cgcgaggccc gcatctggaa gggcgtgttc gagcgcgccg agaaggaggc 900
cggcatcggc cgcggctcca tccgcgccac cgtgctcgtg gagaccctcc cggccgtgtt 960
ccagatggag gagatcctcc acgagctccg cgaccactcc gccggcctca actgcggccg 1020
ctgggactac atcttctcct acgtgaagac cttccgcgcc cgcccggacc gcctcctccc 1080
ggaccgcgcc ctcgtgggca tggcccagca cttcatgcgc tcctactccc acctcctcat 1140
ccagacctgc caccgccgcg gcgtgcacgc catgggcggc atggccgccc agatcccgat 1200
caaggacgac gccgccgcca acgaggccgc cctcgagctc gtgcgcaagg acaagctccg 1260
cgaggtgcgc gccggccacg acggcacctg ggccgcccac ccgggcctca tcccggccat 1320
ccgcgaggtg ttcgagggcc acctcggcgg ccgcccgaac cagatcgacg ccgccgccgg 1380
cgacgccgcc cgcgccggcg tggccgtgac cgaggaggac ctcctccagc cgccgcgcgg 1440
cgcccgcacc gtggagggcc tccgccacaa cacccgcgtg ggcgtgcagt acgtggccgc 1500
ctggctctcc ggctccggct ccgtgccgct ctacaacctc atggaggacg ccgccaccgc 1560
cgagatttcc cgcgtgcaga actggcagtg gctccgccac ggcgccgtgc tcgacgccgg 1620
cggcgtggag gtgcgcgcca ccccggagct cctcgcccgc gtggtggagg aggagatggc 1680
ccgcgtggag gccgaggtgg gcgccgagcg cttccgccgc ggccgctacg ccgaggccgg 1740
ccgcatcttc tcccgccagt gcaccgcccc ggagctcgac gacttcctca ccctcgacgc 1800
ctacaacctc atcgtggtgc accacccggg cgcctcctcc ccgtgcaagc tctaagagct 1860
ctagatcgtt ctgcacaaag tggagtagtc agtcatcgat caggaaccag acaccagact 1920
tttattcata cagtgaagtg aagtgaagtg cagtgcagtg agttgctggt ttttgtacaa 1980
cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 2040
caaaatccag gggatcc 2057
<210> 9
<211> 567
<212> PRT
<213> 未知(Unknown)
<400> 9
Met Ala Thr Asn Ala Ala Ala Pro Pro Cys Pro Cys Tyr Asp Thr Pro
1 5 10 15
Glu Gly Val Asp Ile Leu Gly Arg Tyr Asp Pro Glu Phe Ala Ala Ile
20 25 30
Leu Thr Arg Asp Ala Leu Ala Phe Val Ala Gly Leu Gln Arg Glu Phe
35 40 45
Arg Gly Ala Val Arg Tyr Ala Met Glu Arg Arg Arg Glu Ala Gln Arg
50 55 60
Arg Tyr Asp Ala Gly Glu Leu Pro Arg Phe Asp Pro Ala Thr Arg Pro
65 70 75 80
Val Arg Glu Ala Gly Gly Trp Ala Cys Ala Pro Val Pro Pro Ala Ile
85 90 95
Ala Asp Arg Thr Val Glu Ile Thr Gly Pro Ala Glu Pro Arg Lys Met
100 105 110
Val Ile Asn Ala Leu Asn Ser Gly Ala Lys Val Phe Met Ala Asp Phe
115 120 125
Glu Asp Ala Leu Ser Pro Thr Trp Glu Asn Leu Met Arg Gly Gln Val
130 135 140
Asn Leu Arg Asp Ala Val Ala Gly Thr Ile Thr Tyr Arg Asp Ala Ala
145 150 155 160
Arg Gly Arg Glu Tyr Arg Leu Gly Asp Arg Pro Ala Thr Leu Phe Val
165 170 175
Arg Pro Arg Gly Trp His Leu Pro Glu Ala His Val Leu Val Asp Gly
180 185 190
Glu Pro Ala Ile Gly Cys Leu Val Asp Phe Gly Leu Tyr Phe Phe His
195 200 205
Ser His Ala Ala Phe Arg Ser Gly Gln Gly Ala Gly Phe Gly Pro Phe
210 215 220
Phe Tyr Leu Pro Lys Met Glu His Ser Arg Glu Ala Arg Ile Trp Lys
225 230 235 240
Gly Val Phe Glu Arg Ala Glu Lys Glu Ala Gly Ile Gly Arg Gly Ser
245 250 255
Ile Arg Ala Thr Val Leu Val Glu Thr Leu Pro Ala Val Phe Gln Met
260 265 270
Glu Glu Ile Leu His Glu Leu Arg Asp His Ser Ala Gly Leu Asn Cys
275 280 285
Gly Arg Trp Asp Tyr Ile Phe Ser Tyr Val Lys Thr Phe Arg Ala Arg
290 295 300
Pro Asp Arg Leu Leu Pro Asp Arg Ala Leu Val Gly Met Ala Gln His
305 310 315 320
Phe Met Arg Ser Tyr Ser His Leu Leu Ile Gln Thr Cys His Arg Arg
325 330 335
Gly Val His Ala Met Gly Gly Met Ala Ala Gln Ile Pro Ile Lys Asp
340 345 350
Asp Ala Ala Ala Asn Glu Ala Ala Leu Glu Leu Val Arg Lys Asp Lys
355 360 365
Leu Arg Glu Val Arg Ala Gly His Asp Gly Thr Trp Ala Ala His Pro
370 375 380
Gly Leu Ile Pro Ala Ile Arg Glu Val Phe Glu Gly His Leu Gly Gly
385 390 395 400
Arg Pro Asn Gln Ile Asp Ala Ala Ala Gly Asp Ala Ala Arg Ala Gly
405 410 415
Val Ala Val Thr Glu Glu Asp Leu Leu Gln Pro Pro Arg Gly Ala Arg
420 425 430
Thr Val Glu Gly Leu Arg His Asn Thr Arg Val Gly Val Gln Tyr Val
435 440 445
Ala Ala Trp Leu Ser Gly Ser Gly Ser Val Pro Leu Tyr Asn Leu Met
450 455 460
Glu Asp Ala Ala Thr Ala Glu Ile Ser Arg Val Gln Asn Trp Gln Trp
465 470 475 480
Leu Arg His Gly Ala Val Leu Asp Ala Gly Gly Val Glu Val Arg Ala
485 490 495
Thr Pro Glu Leu Leu Ala Arg Val Val Glu Glu Glu Met Ala Arg Val
500 505 510
Glu Ala Glu Val Gly Ala Glu Arg Phe Arg Arg Gly Arg Tyr Ala Glu
515 520 525
Ala Gly Arg Ile Phe Ser Arg Gln Cys Thr Ala Pro Glu Leu Asp Asp
530 535 540
Phe Leu Thr Leu Asp Ala Tyr Asn Leu Ile Val Val His His Pro Gly
545 550 555 560
Ala Ser Ser Pro Cys Lys Leu
565
<210> 10
<211> 47
<212> PRT
<213> 未知(Unknown)
<400> 10
Met Ala Pro Ser Val Met Ala Ser Ser Ala Thr Thr Val Ala Pro Phe
1 5 10 15
Gln Gly Leu Lys Ser Thr Ala Gly Met Pro Val Ala Arg Arg Ser Gly
20 25 30
Asn Ser Ser Phe Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys
35 40 45
<210> 11
<211> 42
<212> PRT
<213> 未知(Unknown)
<400> 11
Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala
1 5 10 15
Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala
20 25 30
Phe Pro Ala Thr Arg Lys Ala Asn Gly Gly
35 40
<210> 12
<211> 838
<212> DNA
<213> 未知(Unknown)
<400> 12
cgatcggcag gtcatgcgaa atcgcgacga ggctgcgtgc attttgactg attgacgact 60
cacgctgggt aggcccgagg gagaggtggc gcccgcgcct cctcctcctc ctccggcggc 120
ggcggcggcg tggatcgggg cgatgaggcg gtggcgagga ttggcgggag ccatggagat 180
tgccgccgct tggcgggagg aggaggagga ggaggagggg ttgcgtcggc atcggcgggg 240
aggaagcgtg cggaggcggg gcggcgacgt ggcggtggcg gagggcgaaa ggcggcagcg 300
atggctgctg cgtagcgagg caatcatgca gggggaggat gatgatgagg tcgctgccat 360
ctcttctctt ctcttctctt cttctccttc tcctttggcc agcgagagag cagtggcagt 420
gacagtggat gagaagggag ctgggagcag tggcagaggc caggtggaag agaggagatg 480
gcagcgacct catcatcatc ctccccctgc atgattgcct cgctacgcag cagccatcgc 540
tgccgccttt cgccctccgc caccgccacg tcgccgcccc gcctccgcac gcttcctccc 600
cgccgatgcc gacgcaaccc ctcctcctcc tcctcctcct cccgccaagc ggcggcaatc 660
tccatggctc ccgccaatcc tcgccaccgc ctcatcgccc cgatccacgc cgccgccgcc 720
gccggaggag gaggaggagg cgcgggcgcc acctctccct cgggcctacc cagcgtgagt 780
cgtcaatcag tcaaaatgca cgcagcctcg tcgcgatttc gcatgacctg ccgatcgg 838
<210> 13
<211> 491
<212> DNA
<213> 未知(Unknown)
<400> 13
gccgtgacat aaatgacagt ctcagccaac aattgcaagc aaactttgtc taacagcagg 60
aatcgagcaa agaatggatc caaatatacc attcagagca taagcaatgg cacaaaatgg 120
gagagcctcg ggttccttgg cagacaaagc tgctgttcca agcccgtggg cactgaaaaa 180
taaaatttag gtcaagagag taagtaaata accatgcacg cacaaagaaa atgtcattca 240
actattattg catgtttggt atagattaat ttttgagaaa taatcactta ttttacagaa 300
actaatttta tttttcagtg cccacgggct tggaacagca gctttgtctg ccaaggaacc 360
cgaggctctc ccattttgtg ccattgctta tgctctgaat ggtatatttg gatccattct 420
ttgctcgatt cctgctgtta gacaaagttt gcttgcaatt gttggctgag actgtcattt 480
atgtcacggc c 491