CN114703222B - 一种可完全降解多环芳烃植物的培育方法 - Google Patents
一种可完全降解多环芳烃植物的培育方法 Download PDFInfo
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Abstract
一种可完全降解多环芳烃植物的培育方法,按照植物表达模式,对假单胞菌的多环芳烃降解基因簇及红球菌邻苯二酚降解基因簇进行优化重构,分别构建成不同的植物表达载体,然后通过农杆菌介导共转化到同一植物中。利用本发明,使微生物源的两组降解基因簇可在植物中进行表达,获得的转基因植物能够将多环芳烃降解产物转变成乙酰辅酶A,进入三羧酸循环,被植物直接吸收利用,种植这种转基因植物能够提高其降解多环芳烃的能力,有利于修复被多环芳烃污染的土壤环境。
Description
技术领域
本发明属于植物治理土壤污染领域,具体涉及一种可完全降解多环芳烃植物的培育方法。
背景技术
多环芳烃(PAHs)及其衍生物是一类广泛存在于环境中的有机污染物。PAHs大多通过石油、煤等化石燃料以及木材、天然气、有机高分子化合物、纸张、作物秸秆、烟草等含碳氢化合物的物质经不完全燃烧或在还原性气氛中经热分解而生成。
许多PAHs具有显著的急性毒性和潜在的致畸、致癌、致突变作用。多环芳烃化合物由于水溶性差,辛醇-水分配系数高,常被吸附于土壤颗粒上。因此,该类化合物易于从水中分配到生物体内,沉积层中土壤就成为PAHs的主要载体。多环芳烃在环境中不断积累,其半衰期少则2个月、多则几年。多环芳烃进入土壤后,由土壤表面污染进一步导致下层土壤污染,甚至地下水污染。
利用植物治理土壤多环芳烃污染既节能又对环境相对安全。和微生物修复相比,植物修复更适应原地修复,它具有修复面积大、投资少、不破环场地结构、不引起地下水二次污染等优点,在对重金属和有机污染物的处理中已显示明显的有效性,有的已经达到野外应用的水平。但是,由于植物对多环芳烃的羟化作用较弱,因此,对于多环芳烃的降解能力较低。
Peng RH等(Metabolic engineering of arabidopsis for remediation ofdifferent polycyclic aromatic hydrocarbons using a hybrid bacterialdioxygenase complex.Metabolic Engineering,2014,26:100–110)先前的研究表明将萘双加氧酶系统转到植物后可以促进植物吸收多环芳烃,提升植物对多环芳烃的修复能力,然而,Henner P等(Phytotoxicity of ancient gaswork soils.Effect of polycyclicaromatic hydrocarbons(PAHs)on plant germination.Organic Geochemistry,1999,30:275-284)发现,多环芳烃羟化后产物,以及这些产物在植物中的进一步降解产物均可能产生毒性更强的中间产物,使植物的环境适应能力降低。
多环芳烃的降解取决于微生物产生加氧酶的能力,多环芳烃的最初氧化,即苯环加氧是控制PAHs生物降解反应速度的关键步骤。萘双加氧酶(naphthalene dioxygenaseNDO)系统参与萘的羟基化。萘加氧酶系统含有三个组分:铁硫黄素蛋白还原酶NahAa;铁氧还蛋白NahAb和加氧酶,加氧酶由大亚基NahAc和小亚基NahAd组成四聚体,电子从NAD(P)H通过铁硫黄素蛋白还原酶和铁氧还蛋白向加氧酶传送,促进加氧酶将两个氧原子加到萘的一个苯环上。对很多细菌中的NDO基因结构分析发现,萘加氧酶系统多由上述三个组份构成并且种间具有很高的同源性,说明萘双加氧酶系统普遍存在于自然界。
在多环芳烃等诱导下,细菌分泌的双加氧酶把氧加到苯环上,形成C-O键,再经过加氢、脱水等作用使C-C键断裂,苯环数减少,产生水杨醛,水杨醛在水杨醛脱氢酶催化下产生水杨酸,水杨酸可以通过水杨酸羟化酶催化合成邻苯二酚等小分子化合物,全过程需要10个基因参与,分别为NahAa,NahAb,NahAc,NahAd,NahB,NahC,NahD,NahE,NahF,和NahG(Peng RH.Microbial biodegradation of polyaromatic hydrocarbons.FEMSMicrobiology Reviews,2008,32:927–955),然而产生的邻苯二酚对植物具有很强的毒性。
发明内容
本发明提供一种可完全降解多环芳烃植物的培育方法,通过合成生物学方法,对源于微生物的假单胞菌的多环芳烃降解基因簇及红球菌邻苯二酚降解基因簇进行优化重构,分别构建成不同的植物表达载体,再共转化到同一植物中进行表达,将吸收到植物体内的多环芳烃氧化分解成能够进入三羧酸循环的化合物乙酰辅酶A,直接被植物吸收利用,提高植物对多环芳烃的修复能力。
为了达到上述目的,本发明提供如下技术方案:
一种可完全降解多环芳烃植物的培育方法,包括以下步骤:
1)按照植物表达模式,对假单胞菌来源的多环芳烃降解基因簇进行优化重构,构建成包含十价多环芳烃降解基因的由植物启动子控制的多环芳烃降解表达载体,能使多环芳烃降解到邻苯二酚;
2)按照植物表达模式,对微生物来源的邻苯二酚降解基因簇进行优化重构,构建成包含六价邻苯二酚降解基因的由植物启动子控制的邻苯二酚降解表达载体,能使邻苯二酚降解到乙酰辅酶A;
3)将多环芳烃降解表达载体和邻苯二酚降解表达载体通过农杆菌介导共转化到同一植物中进行表达。
进一步,步骤1)及步骤2)中,按照植物偏爱密码、RNA结构稳定性及蛋白翻译效率对多环芳烃降解基因簇和邻苯二酚降解基因簇进行优化重构。
优选地,步骤1)中,所述假单胞菌为恶臭假单胞菌,所述多环芳烃为萘。
又,步骤1)所述多环芳烃降解基因簇来自恶臭假单胞菌的NahAa、NahAb、NahAc、NahAd、NahB、NahC、NahD、NahE、NahF和NahG基因;优化重构后,十价多环芳烃降解基因的具体核苷酸序列依次如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10所示。
优选地,步骤2)中,所述微生物为假单胞菌或红球菌。
又,步骤2)中所述邻苯二酚降解基因簇来自红球菌的CatA、CatB、CatCD、CatI、CatJ和CatF基因;优化重构后,六价邻苯二酚降解基因的具体核苷酸序列依次如SEQ IDNO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQ ID NO.16所示。
进一步,步骤3)中,将构建的多环芳烃降解表达载体和邻苯二酚降解表达载体通过电击法导入根癌农杆菌中,所述根癌农杆菌为为EHA105、LBA4404或GV3101。
又,步骤3)中,所述植物为拟南芥或水稻,转化后利用潮霉素和草胺磷筛选双抗筛选标记植物。
一种用于植物的完全降解多环芳烃的表达单元,其为由一个按植物表达模式优化后的多环芳烃降解基因、组成型启动子CaMV35SΩ启动子和NOS终止子组成的多环芳烃降解基因表达单元,或为由一个按植物表达模式优化后的邻苯二酚降解基因、组成型启动子CaMV35SΩ启动子和NOS终止子组成的邻苯二酚降解基因表达单元;所述多环芳烃降解基因的具体核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示;所述邻苯二酚降解基因的具体核苷酸序列如SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ IDNO.14、SEQ ID NO.15或SEQ ID NO.16所示。
一种用于植物的多环芳烃降解表达载体,其包括10个不同的多环芳烃降解基因表达单元,各多环芳烃降解基因表达单元通过同尾连接酶方法全部连接在植物表达载体上;其中,所述多环芳烃降解基因分别为来自恶臭假单胞菌的NahAa、NahAb、NahAc、NahAd、NahB、NahC、NahD、NahE、NahF和NahG基因按植物表达模式优化后的基因,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点。
一种用于植物的邻苯二酚降解表达载体,其包括6个不同的邻苯二酚降解基因表达单元,各邻苯二酚降解基因表达单元通过同尾连接酶方法全部连接在植物表达载体上;其中,所述邻苯二酚降解基因分别为来自红球菌的CatA、CatB、CatCD、CatI、CatJ和CatF基因按植物表达模式优化后的基因,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点。
又,所述多环芳烃降解表达载体或邻苯二酚降解表达载体中删除了原始表达载体中报告基因的表达盒,并用EcoRI、SalI、BamHI、KpnI、XbaI和HindIII替换了原始表达载体中的多克隆位点片段。
进一步,通过根癌农杆菌将构建的多环芳烃降解植物表达载体和邻苯二酚降解表达载体共转化到拟南芥或水稻中,利用潮霉素和草胺磷筛选双抗筛选标记植物。
本发明中,利用植物降解多环芳烃的重构思路:微生物的多环芳烃降解基因簇只能将多环芳烃降解为邻苯二酚,邻苯二酚的降解需要另一个基因簇完成,该步骤通过邻苯二酚1,2-双加氧酶间位开环裂解邻苯二酚,邻苯二酚的苯环断裂分别生成顺,顺-粘糠酸,之后,在下游粘康酸环异构酶Ⅰ(CatA)、异构酶Ⅰ(CatB)、水解酶(CatCD)、辅酶A转移酶(CatIJ)、乙酰辅酶A乙酰转移酶(CatF)的作用下继续分解,其代谢最终生成琥珀酸盐和乙酰辅酶,进入三羧酸循环,该过程需要7个基因参与,分别为CatA,CatB,CatC,CatD,CatI,CatJ和CatF基因,其中很多微生物的CatC和CatD基因常常合并成1个基因。由于多环芳烃降解的中间产物往往对植物有毒害作用,本发明设计将参与多环芳烃降解和邻苯二酚降解的各个基因进行优化,分别与植物组成型启动子CaMV35SΩ启动子和NOS终止子连接,构建成能够在植物中表达的基因表达单元,通过同尾连接,分别将这些基因表达单元重建成能够将多环芳烃降解到邻苯二酚的植物表达载体及将邻苯二酚降解成乙酰辅酶A的植物表达载体,利用农杆菌介导,通过共转化到同一植物中,使源于微生物的降解基因簇在植物中进行表达,能够将多环芳烃氧化分解成乙酰辅酶A,直接被植物吸收利用,提高植物对多环芳烃的修复能力。
本发明构建降解多环芳烃及邻苯二酚的植物表达载体时,采用同尾限制性酶切克隆法,将EcoRI和XhoI位点被添加到每个基因表达单元的5'域,SalI和HindIII被添加到3'域,再设计引物进行拼接,使各固氮基因的表达单元顺利构建到同一个表达载体上,稳定连接,不易断裂。
与现有技术相比,本发明具有如下有益效果:
本发明重构了假单胞菌的多环芳烃降解基因簇和红球菌邻苯二酚降解基因簇,将两个降解基因簇分别构建成不同的植物表达载体,然后通过农杆菌介导共转化到同一植物中。向植物中转化了重构的微生物源多环芳烃降解途径和邻苯二酚降解途径的植物表达载体后,多环芳烃可以从有毒的化合物转化成能够被植物直接利用的化合物乙酰辅酶A,进入三羧酸循环,被植物直接吸收利用,促进植物生长,种植这种转基因植物能够提高其降解多环芳烃的能力,降低了多环芳烃对环境的污染,修复被多环芳烃污染的土壤环境。
附图说明
图1为本发明实施例1中构建的多环芳烃降解表达载体。
图2为本发明实施例1中构建的邻苯二酚降解表达载体。
图3为本发明实施例2中的转基因拟南芥对多环芳烃萘的抗性对比,其中WT为野生型,含降解表达载体pNah6708和降解表达载体pCat838的为转基因植株。
图4-6为本发明实施例2中转基因植物对多环芳烃萘的降解分析,利用HPLC测定溶液中萘的浓度,其中,纵坐标为相对离子强度,横坐标为滞留时间,图4为初始萘的浓度;图5为野生型拟南芥处理3周后培养液中萘的浓度;图6为转基因拟南芥处理3周后培养液中萘的浓度。
图7为本发明实施例2中同位素分析转基因植物将邻苯二酚转化成三羧酸循环产物-柠檬酸,其中,纵坐标为相对离子强度,横坐标代表不同柠檬酸同位素,m代表非同位素分子质量,m+1代表分子量增加1,m+2代表分子量增加2……依次类推。
图8为本发明实施例3中的转基因水稻对多环芳烃萘的抗性对比,其中WT为野生型,含降解表达载体pNah6708和降解表达载体pCat838的为转基因植株。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
RNA抽提试剂盒RNeasy Plant Mini Kit为德国QIAGEN公司产品(www.qiagen.com),cDNA合成试剂盒购自美国Clontech公司(www.clontech.com);DNA柱回收试剂盒购置美国AmershamPharmacia GE公司(www.amershammedia.com);T/A克隆载体购自大连宝生物公司;KOD FX taq酶购自日本Toyobo公司;各种限制性内切酶和T4 DNALigase均购自上海宝生物工程有限公司Takara。
实施例1重构完全降解多环芳烃的植物表达载体
构建10价基因植物表达载体pNah6708和7价基因植物表达载体pCat838,其具体的构建方法如下:
1、基因设计及全合成
从恶臭假单胞菌(Pseudomonas putida)的基因组序列(GenBank AP013070)中,分别查找获得萘双加氧酶系统4个基因(NahAa、NahAb、NahAc和NahAd)、顺式-二氢二醇萘脱氢酶基因(NahB)、1,2-二氢萘脱氢酶基因(NahC)、2-羟基-苯并吡喃-2-羧酸异构酶基因(NahD)、反式-羟基-苄基丙酮酸水合-醛缩酶基因(NahE)、乙醛脱氢酶基因(NahF)和水杨酸羟化酶基因(NahG)编码的氨基酸序列,进行重构设计。
基于所选基因的氨基酸序列,按照以下原则选择密码子:1)按照植物偏爱密码设计基因,比如从甘氨酸简并密码子选择GCT,亮氨酸简并密码子选择TTG等;2)尽量避开AT重复序列,比如避免从赖氨酸简并密码子选择AAA,避免苯丙氨酸简并密码子选择TTT;3)同一氨基酸尽量避免使用单一密码子,比如精氨酸含有6个简并密码子,以CGT为主,但不能全部选择CGT,也可选择AGA,CGC等在真核生物中使用过的密码子;4)按照密码子优化原则设计后的初步序列还需进一步细化,避免基因中出现RNA转录加尾信号,如AATAAT,AATCAA,AAGCAT等,避免5个以上G+C序列,G+C比例40-60%,避免反向重复序列、茎环结构等,重构设计后的核苷酸序列依次见SEQ ID NO.1-10。
采用PTDS方法对优化重构的上述10个基因进行化学合成,方法参见Ai-shengXiong et al.(PCR-basedaccurate synthesis of long DNA sequences,NatureProtocol,2006,1:791-797和A simple,rapid,high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences.Nucleic AcidsResearch,2004,32(12):e98):首先设计多个60碱基组成的引物,相邻引物之间互为正反引物,引物间设计20个重复碱基,利用PCR进行基因全长扩增,在100μl反应体系中,中间引物的添加量为2ng,外侧引物添加量为中间引物10-15倍,扩增条件为:94℃,预热1min;94℃,30s,50℃,30s,72℃,10min,使用的Taq DNA聚合酶为KOD FX taq酶(Toyobo公司,日本),共25个循环。
PCR结束后,1%琼脂糖胶回收,取10μl直接与T/A克隆载体相连,4℃连接过夜,获得质粒T2,将质粒T2高效转化到大肠杆菌DH5α感受态细胞中,获得阳性克隆,通过序列测定鉴定筛选正确的基因。
2、构建将多环芳烃降解到邻苯二酚的植物表达载体
将用于多环芳烃降解到邻苯二酚的优化重构后的10个基因NahAa、NahAb、NahAc、NahAd、NahB、NahC、NahD、NahE、NahF和NahG通过“改良重叠延伸PCR技术”与含有CaMV35S启动子和NOS终止子的pYPX245(Genbank AY178049.1)载体连接,方法参见Peng RH et al.(Adirect and efficient PAGE-mediated overlap extension PCR method for genemultiple-site mutagenesis.Applied Microbiology Biotechnology,2006,73:234-40),构建基因表达单元,序列测定获得所有10个基因的植物表达单元,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点,将所有表达单元酶切后通过同尾连接酶方法全部连接至改造的pCAMBIA1301植物表达载体,删除了原始表达载体中报告基因的表达盒,并用EcoRI、SalI、BamHI、KpnI、XbaI和HindIII替换了原始表达载体中的多克隆位点片段,每插入一个表达单元均进行质粒抽提和酶切鉴定,对鉴定正确的中间载体进行核苷酸全序列分析测定,最终构建成十价多环芳烃降解基因植物表达载体pNah6708(图1)。
3、构建将邻苯二酚降解到乙酰辅酶A的植物表达载体
将邻苯二酚降解到乙酰辅酶A的6个基因CatA、CatB、CatCD、CatI、CatJ和CatF通过“改良重叠延伸PCR技术”与含有CaMV35S启动子和NOS终止子的pYPX245(GenbankAY178049.1)载体连接,构建基因表达单元,序列测定获得所有6个基因的植物表达单元,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点。将所有表达单元酶切后通过同尾连接酶方法全部连接至改造的pBI121植物表达载体,删除了原始表达载体中报告基因的表达盒,并用EcoRI、SalI、BamHI、KpnI、XbaI和HindIII替换了原始表达载体中的多克隆位点片段,表达载体中的新霉素抗性基因NptII替换为草胺磷抗性基因bar,每插入一个表达单元均进行质粒抽提和酶切鉴定,对鉴定正确的中间载体进行核苷酸全序列分析测定,最终构建成六价邻苯二酚降解基因植物表达载体pCat838(图2)。
实施例2转基因的拟南芥完全降解多环芳烃
1、制备双载体农杆菌
农杆菌菌株为根癌农杆菌EHA105、LBA4404或GV3101菌株(BiovectorCo.,LTD)。
挑取单菌在25ml YEB培养基中培养过夜,取5ml菌液转接到100ml YEB培养基,所述YEB培养基中添加有利福平50mg/l,培养至OD600=0.7-0.8,菌液冰上放置10分钟,5000rpm离心10min,4℃,收集菌体,加入100ml无菌双蒸水清洗两次。加入4ml 10%甘油悬浮菌体,转到50ml离心管。5500rpm离心10min,4℃。收集菌体,加入500μl 10%甘油悬浮菌体,转到1.5ml离心管。
取70μl农杆菌感受态细胞,加入1μl十价多环芳烃降解基因植物表达载体pNah6708和1μl六价邻苯二酚降解基因植物表达载体pCat838,混匀,转到0.1cm电击杯中进行电击,电击参数:200Ω,1.7KV,2.5F,电击后立即加入800μl SOC培养液培养,所述SOC培养液中含有:2%(W/V)Tryptone,0.5%(W/V)Yeast Extract,0.05%(W/V)NaCl,2.5mMKCl,10mM MgCl2和20mM glucose,培养1小时后,取100μl涂15μg/L卡那霉素和15μg/L四环素抗性板筛选含有两种降解表达载体的农杆菌转化子,28℃培养。
2、粘花法转化拟南芥
挑取上述步骤1获得的抗卡那霉素和四环素的农杆菌,单菌落接菌在5ml含对应卡那霉素和四环素抗生素的LB培养基中28℃培养2天,将5ml菌液转到500mL的LB液体培养基中28℃培养16-24小时(OD=1.5-2.0)。
室温下离心收集菌体,4000g离心10分钟,用等体积5%的新鲜蔗糖溶液悬浮,加入0.02%的Silwet-77混匀后转移到烧杯中,每个菌株用300毫升转化,转2-3钵,隔7天后再转化1次。
转化操作中将拟南芥植株倒置后浸入菌液中10秒钟,莲座和花序都要侵染,侵染后将转化植株菌液空干3-5秒。用保鲜膜将转化植株圈好,平放16-24小时,转化后不要放置在高温和强光下。揭开保鲜膜,保持一定湿度,再生长1个月后收种子,利用30μg/mL潮霉素和5mg/L草胺磷进行转化植株筛选。
3、验证转基因拟南芥具有16个基因的表达产物
将拟南芥幼苗液氮冷冻后抽提总RNA,采用RNeasy Plant Mini Kit试剂盒提取抽提转基因拟南芥总RNA,取约50μl拟南芥总RNA进行cDNA合成,利用cDNA合成试剂盒按照说明书合成cDNA第一链。
针对合成基因的具体核苷酸序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ IDNO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQID NO.16,设计专一性引物。
取1μl cDNA合成产物,检测每个基因的表达,PCR扩增条件为:94℃,预热1min;94℃,30s,50℃,30s,72℃,30s,扩增35循环,能在转基因植物中检测到专一性条带,而野生型拟南芥没有,表明每个基因都能够转录表达。
4、转基因拟南芥对多环芳烃的耐受性和降解
将转基因拟南芥自交纯合3代,获得纯合转化株,收取种子,播种后,移栽到含有萘100mg/L的基本培养基中培养3周,观察植物的生长情况,参见图3,转基因拟南芥对多环芳烃的耐受性明显强于对照植株。
收集培养过观察植株的培养基,用总体积为60mL体积比为1:1的丙酮和正己烷溶液分3次萃取,每次20mL并在超声水浴中超声萃取30分钟,然后将萃取液收集,利用旋转蒸发仪浓缩至干,加入2mL甲醇溶解,将抽提液进行HPLC分析获得萘含量的色谱图,流动相为甲醇-水,流速为1mL/min,柱温30℃,进样量为40μL,在此条件下所得萘的色谱图清晰(参见图4-6及表1)。
表1
编号 | 萘(mg/kg培养基) | 降解率(%) |
起始浓度 | 89.0(±1.15) | / |
野生型对照 | 64.1(±1.16) | 28.1 |
转基因拟南芥1 | 44.1(±1.25) | 50.5 |
转基因拟南芥2 | 45.8(±1.76) | 48.5 |
转基因拟南芥3 | 33.3(±1.61) | 62.6 |
图4-6及表1结果表明:转基因拟南芥培养3周后降解培养基中的萘为48.5-62.6%,而野生型拟南芥对照只有28.1%。
利用稳定性同位素邻苯二酚饲喂转基因拟南芥并进行质谱分析(图7),质谱分析表明,转基因植物能够将邻苯二酚进一步转化成三羧酸循环产物-柠檬酸。
实施例3转基因水稻完全降解多环芳烃
1、农杆菌介导转化水稻
利用实施例1中获得的含有两个降解表达载体pNah6708和pCat838的农杆菌菌株对水稻愈伤组织进行转化:
以N6培养基为基本培养基,将去壳的水稻种子,授粉后12-15天的幼胚,经表面消毒后接种到N6D2培养基中诱导愈伤组织,其中,N6培养基中含有水解乳蛋白500mg/L、蔗糖30g/L、2,4-D 2mg/L和植物凝胶2.5g/L,pH5.8,培养4-7天后取愈伤组织进行转化。
将含有降解表达载体pNah6708和pCat838的农杆菌培养至OD0.8-1.0后,5000g离心8分钟,重整水清洗一次,等体积MS培养液悬浮侵染愈伤组织8分钟后,吸干放置在含MS培养基、NAA 1mg/L和BA2mg/L的培养基中,22℃共培养3天。
之后转入筛选培养基,所述筛选培养基中包括N6培养基、脯氨酸3g/L、水解酪蛋白0.6g/L、蔗糖30g/L、2,4D0.3μg/mL、植物凝胶4g/L、头孢Cb500μg/mL、潮霉素HAT30μg/mL和草胺磷10μg/mL,转化后的愈伤组织在含有潮霉素30μg/mL和草胺磷10μg/mL抗性的筛选培养基上培养3~4代,转入分化培养基中,所述分化培养基为MS培养基,其中添加KT2mg/L、潮霉素30μg/mL和草胺磷2μg/mL;幼芽长至2mm转移到生根培养基,所述生根培养基中含1/2MS培养基和IBA0.5 mg/L;以上分化和生根培养基中分别加入酶水解乳蛋白(CH)500mg/L、谷氨酰胺或精氨酸0~700mg/L、蔗糖30~80g/L和琼脂6g/L,pH 5.8。筛选培养基的继代周期为25天,将淡黄色的胚性愈伤组织转入分化培养基中,30天左右分化出芽,光照强度1500~2000lx,12~14h/d。
2、验证转基因水稻中外源基因的表达产物。
将水稻幼苗液氮冷冻后,用RNA抽提试剂盒RNeasy Plant Mini Kit抽提转基因水稻总RNA,利用cDNA合成试剂盒合成cDNA第一链。
取约50μl水稻总RNA进行cDNA合成,按Clontech公司的cDNA合成说明书操作进行第一链合成。
按照核苷酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ IDNO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQ ID NO.16所示合成基因的核苷酸序列设计专一性引物。
取1μl cDNA合成产物检测每个基因的表达。PCR扩增条件为:94℃,预热1min;94℃,30s,50℃,30s,72℃,30s,扩增35循环,能在转基因植物中检测到专一性条带,而野生型水稻没有,表明每个基因都能够转录表达。
3、转基因水稻完全降解多环芳烃
将基因水稻自交纯合3代,获得纯合转化株,收取种子。播种后,移栽到含有100mg/L萘的基本培养基中,培养3周,观察水稻的生长情况,参见图8,可见转基因水稻对多环芳烃的耐受性明显强于对照植株。收集所有的培养基,用60mL 1:1的丙酮和正己烷溶液分3次萃取,每次20mL并在超声水浴中超声萃取30分钟,然后将萃取液收集,利用旋转蒸发仪浓缩至干,加入2mL甲醇溶解。将抽提液进行HPLC分析,流动相为甲醇-水,流速为1mL/min,柱温30℃,进样量为40μL,在此条件下所得萘的色谱图清晰,检测结果参见表2。
表2
编号 | 萘(mg/kg培养基) | 降解率(%) |
起始浓度 | 89.0(±1.15) | / |
野生型对照 | 51.9(±2.83) | 41.6 |
转基因水稻1 | 15.3(±1.78) | 82.8 |
转基因水稻2 | 17.4(±2.03) | 80.5 |
转基因水稻3 | 10.4(±1.55) | 88.3 |
检测结果表明:转基因水稻培养3周后对培养基中萘的降解率为80.5-88.3%,而水稻初始对照品种的萘降解率只有41.6%。
序列表
<110> 上海市农业科学院
<120> 一种可完全降解多环芳烃植物的培育方法
<130> 2211029
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 987
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggaacttc tcatacagcc aaacaatcgc ctcattagct ttagtcccgg cgccaacctt 60
ctggaagtgc ttcgcgaaaa cggtgtcgct atttcctaca gttgtatgtc tgggcgttgc 120
ggaacctgcc gctgtcgggt tacagatggc agtgtaattg attctggtgc tggatctggg 180
ttgccaaacc tcgtggacga gcattatgtg ctcgcctgtc agtcagtact tactcacaat 240
tgcgcgatcg aaatcccaga aaccgacgaa atcgtcaccc acccggcgag aatcatcaag 300
ggcactgtgg tcgccgtcga gtcgcccact cacgatatcc gtcgcctacg cgtacgcctc 360
gctaagccct tcgagttctc acccggacag tacgcgacat tgcagttcag tcctgagcat 420
gcgcgtccgt attcaatggc aggtctgcca gatgaccaag aaatggagtt ccacatacgc 480
aaggtgccgg gtgggcgcgt aacggagtat gttttcgagc acgtccgcga aggtacaagc 540
atcaagttga gcgggccact tggtacggct tatttgcgtc agaaccacac cgggccgatg 600
ctctgtgtgg gcggtgggac cggactagca ccggtgctgt cgattgttcg cggcgcgctg 660
aagttgggta tgacaaaccc catcctcctt tatttcggag tgcgcagtca gcaagacctc 720
tacgacgcag agcgattgca caaactcgcc gctgatcacc ctcaactgac cgtacacacg 780
gtaatcgcaa tgggcccgat taatgagagt cagcgagccg gtctagttac cgatgtgatc 840
gaaaaagaca tcatttcgct ggctgggtgg agggcctacc tgtgcggcgc accagcgatg 900
gttgaagcgc tttgcaccgt taccaagcat cttggaatat cacccgaaca tatttatgcc 960
gatgccttct atcctggtgg aatctga 987
<210> 2
<211> 324
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgacagaaa aatggattga agcagtcgcc ctttctgaca tcccagaagg tgatgtcctc 60
ggcgtgactg tcgagggcaa ggagctggcg ttgtacgaag tggaaggcga aatctacgct 120
accgacaacc tgtgcacgca tggtgctgct cgtatgtctg atggttacct cgaaggtcgt 180
gaaatcgaat gtcctttgca tcaaggtcgt ttcgacgtct gtacaggcag agctctctgt 240
gctcctgtca ctgagaacat caaaacctac gcagtcaaga tcgagaacct gcgtgtcatg 300
atcgacttgt ctggagagtt ctaa 324
<210> 3
<211> 1350
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaactaca aaaacaaaat cttggtgtct gaatctggtc tgactcaaaa gcacctgatt 60
catggtgatg aagaattgtt ccagcacgaa ctgagaacca tctttgctcg taactggttg 120
ttcttgactc atgactcttt gattccatct ccaggtgact atgttaccgc aaagatgggt 180
attgacgagg tcatcgtctc tcgtcagtct gacggttcga ttcgtgcctt cctgaacgtt 240
tgtcgtcacc gtggcaagac gttggttaac gctgaagctg gcaatgccaa aggtttcgtt 300
tgctcttacc atggttgggg tttcggttcc aacggtgaat tgcagtctgt tccattcgaa 360
aaggagttgt acggtgagtc gctcaacaag aagtgtttgg gtttgaaaga agtcgctcgc 420
gtggagtctt tccatggttt catctatggt tgcttcgatc aggaggctcc tcctttgatg 480
gactatctgg gtgacgctgc ttggtacttg gagcctatct tcaaacattc aggtggtttg 540
gaactggtcg gtcctccagg caaggttgtt atcaaggcta actggaaggc accagctgaa 600
aactttgtgg gggatgcata ccacgtgggt tggacgcacg cgtcttcttt gcgttctgga 660
gagtctatct tcgcgtctct cgctggcaac gcagtgctgc cacctgaagg tgcaggtttg 720
caaatgacct ccaagtacgg ttctggcatg ggtgtgttgt gggacggata ttctggtgtg 780
cattctgcag acttggttcc agagttgatg gcattcggtg gttctaagca ggaaaggttg 840
aacaaagaaa ttggtgatgt tcgtgctcgg atctatcgtt ctcacctcaa ctgcaccgtt 900
ttcccaaaca acagcatgct gacctgctct ggtgttttca aagtatggaa cccgatcgac 960
gcaaacacca ccgaggtctg gacctacgcc attgtcgaga aagacatgcc agaggatttg 1020
aagcgtcgtt tggcagactc ggttcagcgt acgtttgggc ctgctggctt ctgggaatct 1080
gacgacaatg acaatatgga aacagcgtcg caaaacggca agaaatatca atcccgtgat 1140
tctgatttgt tgtcaaacct tggtttcggt aaggacgtat acggtgacgc ggtctatcct 1200
ggtgttgttg gcaagtctgc aatcggtgag acctcttatc gtggtttcta ccgtgcttac 1260
caggcacacg tttcttcttc caactgggct gagttcgagg atgcctcttc tacttggcat 1320
accgaattga cgaagactac tgatcgttaa 1350
<210> 4
<211> 585
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgatgatca acatccaaga agacaagctg gtctccgccc acgacgccca agagtttctt 60
cgtttcttca attgccacga cgcggctttg caacaagaag ccaccacgct gctgaaccgg 120
gaagcgcatc tgttggacat tcaggcttac cgggcttggt tagagcactg cgtggggtca 180
gaggttcaat atcaggtcat ttcacgcgaa ctgcgcgccg cttccgagcg ccgttataag 240
ctcaatgaag ccatgaacgt ttacaacgaa aattttcagc aactgaaagt tcgaatcgag 300
catcaacttg atccgcaaaa ctggagcaac agcccgaagc tgcgctttac tcgcttcatc 360
accaatgtcc aggccgcaag ggacgtagat gacgaagagc tacttcacat ccgctccaac 420
gtcattctgc accgggcacg acgtggcaat caggtcgatg tcttctacgc cgcccgggaa 480
gacaaatgga aacgtggcga aggtggagtg cgaaaattgg tccagcgatt cgtggattac 540
ccagagcgca tacttcagac gcacaatctg atggtctttc tgtga 585
<210> 5
<211> 789
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgggtaatc aacaagttgt gtcaatcact ggtgctggtt ctggtatcgg tcttgaactg 60
gttcgttcat tcaagttggc tggttactgt gtgtctgcac ttgttcgtaa cgaggaacaa 120
gaggcacttc tttgcaacga gttcaaggat gcactggaga tcgttgttgg tgatgttcgt 180
gatcatgcaa ctaacgagaa gctgatcaag cagactattg atcgtttcgg tcatctggac 240
tgcttcatcg caaacgctgg tatctgggac tacatgctga acatcgaaga accttgggag 300
aaaatctcat catcattcga tgaaatcttc gacatcaacg tgaagtcata cttctctggt 360
atctctgctg cactgccaga gctgaagaag actaacggtt ctgtggtgat gactgcatca 420
gtgtcatcac atgcagttgg tggtggtggt tcatgctaca tcgcatctaa gcacgcagtg 480
ctgggtatgg tgaaggcact tgcatacgaa cttgcacctg agattcgtgt gaacgctgtg 540
tcacctggtg gtactgtgac ttcactgtgt ggtcctgcat ctgctggttt cgacaagatg 600
cacatgaagg atatgcctgg tatcgacgac atgatcaagg gtctgactcc acttggtttc 660
gctgctaagc ctgaagacgt ggtggcacca tacctgttgc tggcatcacg taagcaaggt 720
aagttcatca ctggtactgt gatctcaatc gacggtggta tggcactggg tcgtaagtaa 780
ctagcataa 789
<210> 6
<211> 909
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgtcaaagc aagctgctgt gatcgaactt ggttacatgg gtatctctgt gaaggaccct 60
gatgcatgga agtcattcgc aatgaacatg cttggtctgc aagttctgga tgagggtgag 120
aaggatcgtt tctaccttcg tatggactac tggcatcatc gtatcgtggt tcatcattct 180
gctgaggatg acttggagta ccttggttgg cgtgttgctg gtaagcctga gttcgaagca 240
cttggtcaga agctgatcga tgctggttac aagatccgtg tgtgtgacaa ggttgaggca 300
caagaacgta tggtgttggg tctgatgaag actgaggacc ctggtggtaa tccaactgaa 360
atcttctggg gtccacgtat cgacatgtct aacccattcc accctggtcg tccactgcat 420
ggtaagttcg tgactggtga tcaaggtctt ggtcactgca tcgttcgtca gactgatgtt 480
gcagcagcac acaagttcta ctcactgctg ggtttccgtg gtgatgtgga gtatcgtatc 540
ccactgccaa acggtatgac tgctgaactg tcattcatgc actgcaacgc acgtgatcat 600
tcaatcgcat tcggtgcaat gccagcagca aagcgtctga accatctgat gcttgagtac 660
actcacatgg aagacttggg ttacactcac caacagttcg tgaagaacga gatcgacatc 720
gcacttcagc ttggtatcca tgctaacgac aaggcactga ctttctatgg tgcaactcca 780
tctggttggc tgatcgaacc aggttggcgt ggtgcaactg caatcgatga agctgagtac 840
tacgttggtg acatcttcgg tcatggtgtt gaagcacctg gttacggtct ggatgtgaag 900
ctgtcataa 909
<210> 7
<211> 612
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgattgttg acttctactt cgatttcttg tcaccattct catacttggc aaaccaacgt 60
ctgtcaaaac tggcacaaga ctacggtctt actatccgtt acaacgcaat cgaccttgca 120
cgtgtgaaga tcgcaatcgg taacgttggt ccatcaaacc gtgacttgaa ggtgaaactg 180
gactacttga aggtggatct gcaacgttgg gcacagctgt acggtatccc actggtgttc 240
cctgcaaact acaactcacg tcgtatgaac atcggtttct actactctgg tgctgaagca 300
caggcagcag catacgtgaa cgtggtgttc aacgcagtgt ggggtgaagg tatcgcacct 360
gatctggagt cactgcctgc actggtgtct gagaagctgg gttgggatcg ttctgcattc 420
gagcatttcc tgtcatcaaa cgcagcaact gagcgttacg atgaacagac tcatgctgca 480
atcgaacgta aggtgttcgg tgtgccaact atgttccttg gtgatgagat gtggtggggt 540
aacgatcgtc tgttcatgct ggaatctgca atgggacgtc tttgtcgtca gaacgctgac 600
ttgtcatcat aa 612
<210> 8
<211> 1008
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgggtttga acaaggtgat caaaactact cgtcttactg ctgaagacat caatggtgca 60
tggactatca tgccaactcc atcaactcct gatgcatctg actggcgttc aactaacact 120
gtggacttgg atgagactgc acgtatcgtt gaagaactga tcgctgctgg tgtgaatggt 180
atcttgtcaa tgggtacttt cggtgaatgt gcaactctta cttgggagga gaaacgtgat 240
tacgtgtcaa ctgttgtgga gactatccgt ggtcgtgtgc catacttctg tggtactact 300
gcactgaaca ctcgtgaagt gattcgtcag actcgtgaac tgatcgacat cggtgctaac 360
ggtactatgc ttggtgtgcc aatgtgggtg aagatggatc ttccaactgc tgtgcagttc 420
taccgtgatg tggcaggtgc tgttcctgag gctgcaatcg caatctacgc taaccctgaa 480
gcattcaagt tcgactttcc tcgtccattc tgggctgaga tgtctaagat cccacaggtg 540
gtgactgcaa agtaccttgg tatcggtatg ctggacctgg acctgaagtt ggcaccaaac 600
atccgtttcc tgccacatga ggatgactac tacgcagcag cacgtatcaa ccctgaacgt 660
atcactgcat tctggtcatc tggtgcaatg tgtggtcctg caactgcaat catgttgcgt 720
gacgaagtgg aacgtgctaa gtcaactggt gattggatca aggctaaggc tatctctgac 780
gacatgcgtg ctgctgactc aactcttttc ccacgtggtg acttctctga gttctctaag 840
tacaacattg gtcttgagaa ggcacgtatg gatgctgctg gttggctgaa ggctggacca 900
tgtcgtccac catacaacct tgtgcctgaa gactacctgg ttggtgcaca gaagtctggt 960
aaggcatggg cagcacttca tgcaaagtac tcaaagtaac tagcataa 1008
<210> 9
<211> 1464
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgggtaaga ctaagctgtt catcaacaat gcatggatcg actcatctga tcaacagact 60
ttcgaacgta agcatcctgt gtcatctgag gtgatgactg agtctgcaaa cgcaactgtg 120
actgatgcaa tcaaggctgc acaagctgct gaggaggcat tcaagacttg gaaggatgtt 180
ggtccatctg agcgtcgtcg tcttcttctg aaggttgctg atgtgatgga gtctaagact 240
cctaagttca tcgaagtgat ggcaatggag gttggtgcat ctgcactgtg ggctggtttc 300
aacgttcatg catctgcaaa cgtgttccgt gaggctgcat cacttgctac tcagatccag 360
ggtgagacta tcccaactga caaagctgag actctgtcaa tgactctgcg tcaaccagtg 420
ggtccaatcc tgtcaatcgt gccttggaat ggtactgctg tgctggcagc acgtgcaatc 480
gcatacccac tggtgtgtgg taacactgtt gtgttcaagg gttctgagtt ctcaccagca 540
actcatgcac tgatcactca gtgtgtgcag gaagcaggtc ttcctgctgg tgtgctgaac 600
tacctgaact catcaccaga tcgttcacca gagatcgcag atgcactgat ctcagctaag 660
gagattcgtc gtatcaactt cactggttca actcgtgtgg gttcaatcat cgcacagaag 720
gcagcacaac atctgaagcg ttgtctgctg gaacttggtg gtaagtcacc actgatcgtg 780
ttggatgatg ctgacatcaa cgcagcagtg aaggcagcag tgttcggttc atttctgttc 840
caaggtcaaa tctgcatgtc aactgagcgt ttggtggttg atgagaagat tgctgacgag 900
ttcgtggcac gtttcgtgga gaagactgaa cgtctgtctg ttggtgatcc atgtctgact 960
ggtgactgta tcattggtcc tatggtgtca ccaaactcag gtgagcgtat caatggtctg 1020
ttcaaggatg caatcgacaa gggtgcaaag gtggtgtgtg gtggtatggc acagggtgca 1080
gtgatgccag caactatcct ggatcatgtg aagtctgaca tgcgtatcta cgatgaggaa 1140
actttcggtc caatcactgt ggtgatccgt tgcaagggtg aagctgaggc aatccgtatc 1200
gcaaacgact ctgtgtacgg tctgtcatca ggtgtgttcg gtcgtgacat caaccgtgca 1260
ctgcgtgtgg gtatgtcaat cgagtacggt tgtgtgcata tcaacggttc aactgttcag 1320
aacgaagcac aggcaccata cggtggtact aagaacactg gttacggtcg tttcgatggt 1380
cgtgctgtga tcgatgagtt cactgagctg aagtggctga ctatcgaacc attcgaacaa 1440
caatacccat tctaactagc ataa 1464
<210> 10
<211> 1308
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgggtaaga acaacaaact tggtcttcgt atcggtattg ttggtggtgg tatctctggt 60
gttgcacttg cactggaact gtgtcgttac tcacatatcc aggttcagct gttcgaagct 120
gcacctgcat tcggtgaagt tggtgctggt gtgtcattcg gtccaaacgc tgttcgtgca 180
atcgttggtc ttggtcttgg tgaagcatac ctgcaagtgg ctgatcgtac ttctgaacct 240
tgggaagatg tgtggttcga gtggcgtcgt ggttctgacg catcatacct tggtgcaact 300
atcgcacctg gtgttggtca gtcatctgtg catcgtgctg acttcatcga cgcacttgtg 360
actcatctgc ctgaaggtat cgcacagttc ggtaagcgtg caactcaagt ggaacagcag 420
ggtggtgaag tgcaggtgct gttcactgat ggtactgagt accgttgtga tctgctgatc 480
ggtgctgatg gtatcaagtc tgcactgcgt tcacatgttc ttgaaggtca aggtcttgca 540
ccacaagttc cacgtttctc tggtacttgt gcataccgtg gtatggtgga ctcactgcat 600
ctgcgtgaag cataccgtgc acatggtatc gatgaacatc tggtggatgt tccacagatg 660
taccttggtc ttgatggtca catcctgact ttccctgttc gtaacggtgg tatcatcaac 720
gtggttgcat tcatctctga tcgttctgaa cctaagccaa cttggcctgc tgatgcacct 780
tgggttcgtg aggcatcaca acgtgagatg ctggatgcat tcgcaggttg gggtgatgca 840
gcacgtgcac tgctggagtg cattcctgca ccaactctgt gggcactgca tgaccttgca 900
gaactgcctg gttacgtgca tggtcgtgtt gtgctgatcg gtgatgcagc acatgcaatg 960
ctgccacatc aaggtgctgg tgctggtcaa ggtcttgagg acgcatactt ccttgccaga 1020
ctgttgggtg atactcaagc tgatgctggc aaccttgctg agctgcttga agcctacgat 1080
gacctgcgtc gtccacgtgc atgtcgtgtg caacagactt catgggagac tggtgaactg 1140
tacgagcttc gtgatcctgt ggttggtgca aacgaacaac tgctgggtga gaaccttgca 1200
actcgtttcg actggctgtg gaaccatgat ctggatactg accttgctga ggcacgtgca 1260
cgtcttggtt gggagcatgg tggtggtggt gcactgcgtc aaggttaa 1308
<210> 11
<211> 852
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgactacta tggagaatcc aactgcacat ggttctggta atgctgcaac tgataagttc 60
aagtctgagc gtgtgacttc tgatacttct gtggaacgtg catctgcaat ctacaaggat 120
cttctggatg cactggctgg tatcgttgac aagcatcaag tgacttacga tgagtaccgt 180
gttctgaagc agtggctgat cgatgttggt gagtatggtg aatggccact gtggctggat 240
gtgttcctgg aacacgagat cgagaaggtg cattacaacc gtaagggttt cactggtact 300
aagggttcaa tcgaaggtcc atactacgtt cctgactcac ctaagctgcc atcaaagtgt 360
actatgccaa tgcgtgagaa ggataaggtt gcaccaccac tggtgttcaa gggtcaggtg 420
actgacctgg aaggtaacgg tcttcctggt gcaactgtgg aactgtggca cgctgatgaa 480
gaaggtttct actcacagtt cgcacctggt atccctgaat ggaacctgcg tggtactgtt 540
gaggttgatg agaacggtaa cttcgagatc actactcttg aacctgcacc ataccagatc 600
ccatctgacg gtccaactgg ttggttcatc aagtcatacg gtggtcatcc ttggcgtcct 660
gcacatctgc atctgatggt gaaggcacct ggtaagcgta ctatcactac tcagctgtac 720
ttccaaggtg gtgaatgggt tgaagatgat gttgcaactg ctgtgaagcc tgaactgatc 780
ctggacccac aacctaacgc tgatggtgtt gctgaggtga cttacaactt cgtgcttgat 840
cctgaagcat aa 852
<210> 12
<211> 1128
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgtctgatc ctgatcttaa gattgcatct gtgactacta ctatcatcga tgttccactg 60
atccgtccac ataagtttgc tactactact tctgaggcac aaccaatcct gcttgttgct 120
gtgactactg agaacggtgt gactggttat ggtgaaggtg ttgttcctgg tggtccttgg 180
tggggtggtg agtctgttga gactatgcag cagatcgttg atcgttacat cggtccatac 240
atcattggtc gtggtgttga tgaaatctct ggtgtgatgg ttgacatcga acgtatcgtt 300
gctaacgcac gtttcgcaaa ggctgctgtt gatgttgcta tgcatgatgc atggggtcgt 360
gcactgggtg ttcctgttgc atcactgctt ggtggtgcat tccgtactgg tgttgatgtt 420
cgttgggcac tgggtgctgc accactggag gagatcgtgg aagaggtgac tcataagcgt 480
gaacaacgtc tgaacttctc attcaagctg aagatgggtg cactggaccc tgctgtggat 540
actggtcgtg tggtgaagat cgtggaagca ttcggtggtg aggttggttt ctcaatcgat 600
gtgaatgcac gttgggatcg tttcactgca cttcgtcatg tgccacagct tgttgacggt 660
ggtgtggaac tgatcgaaca accaactcct gctgatcaac tggatgttct tgctgagatc 720
aaccgtcgtg tgtctgcacc tgtgatggct gatgagtctg tgcagactcc acatgatgca 780
tacgaggttg caaagctggg tgctgctgat gttgtggcac tgaagactac taagtgtggt 840
ggtcttcagc gttctaagca ggtggttgct gttgctaagg ctgctggtct gcgttgtcat 900
ggtgctactt ctatcgaagg tccaatcggt actgctgcat caatccattt cgcatgtgct 960
gaacctggta tcgactatgg tactgaactg ttcggtccac agctgttcgc tgtggaactg 1020
cttcagaagc cactggacta ctctgaaggt caggtgcatc ttcctgaagg tcctggtctg 1080
ggtgttgatc tggacatgga cgtggtgaag aagtatgcac gttcataa 1128
<210> 13
<211> 849
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atgatcgaag cacgttacct tgaaatcgat ggtgcacttg gtttcgttga gatcatcact 60
cctgataacc aacctgcaac tgaagcacca actgttctgt gtctgcatac tgctggtcag 120
tctggtgttc agtggcgtca tgttgcatct gaactggctg atcgtggtta tcgtgtgatc 180
gttcctgatc ttcctggtca tggtcgttca gaacctgctg ttgatggtcc tgttacttct 240
atcgttgcat tcggtgactg gcttggtaag gtgcttgatg cacttgctgt tgaacgtcca 300
ttcgttgttg gttgctcaat cggtggtaag ctgactctgg aacttgcaac tcgttcatca 360
cgtccacttg ctggtgctat cgcaatggct gctgaagcag gtccaggtcg tgtgaacgtt 420
ggtggtctgc gtcgtgaact ggaagatgtt gcaggtccat cacgtgctga tcgtacttac 480
ctgggtactc tggcatctgt gggtcatgca gttcctgaag caaaggcaca tctgatcgca 540
actatgcata agcgtgaaga ccctgaaatc tcatcatctg atctgatcgg ttggggtact 600
catgatgttc gtgatcgtat cgctgagatc acttgcccac tgcgtcttgt tgctggtgca 660
caggaccctt ggatcgacgc tggtcgtgtt gaacagtctg tgtctgcact ggatgctgca 720
cgtcctggtc ttgcatcatt cactctgctt gaaggtatcg gtcactaccc tatggaggag 780
atcgacgact tcgcatctgt ggctgatgag tggctgactg aacttcgtgc tgctgtggtg 840
ctgtcataa 849
<210> 14
<211> 750
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggtgaaca aggtgttcgc aactgctgct gaggctgttg ctgatgttcc tgacggtgca 60
tcacttgctg ttggtggctt tggactcgtg ggaatcccat ctgtcctgat cgatgcactg 120
cttgcacaag gtgcaactga tctggagact gtgtccaaca actgtggaac tgacggcttc 180
ggactgggac tgctgcttga gcagcatcgt atccgtcgta ccatctcatc ctacgttggt 240
gccaacaagg agttcgcacg tcagtacctg tctggtgagc tggaggttga gctgactcca 300
cagggaaccc ttgctgagcg tatgcgtgct ggaggagcag gcatccctgc cttctacact 360
cctgctggtg ttggaactca ggttgctgat ggtggactgc caatccgtta cgatggacaa 420
ggaggcatcg ctgttgcatc caagcctaag gagactcgtg agttcgatgg tgaggcattc 480
gttcttgaac gtgccatccg taccgacttc gcactggttc atgcatggaa gggtgatcgt 540
cttggcaacc ttgtgtatcg tgagactgca cgtaacttca atcctgatgc tgctggtgct 600
ggacgtatca ccatcgcaca ggttgagtac cttgttgaac ctggtgagat cgatcctgct 660
gaggtccaca ctcctggcat cttcgttcag cgtgttgtcg aggttggcaa gcaggagact 720
ggcatcgaga accgtactgt tcgtgcataa 750
<210> 15
<211> 642
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgtcatgga ctcgtgatga gatggcagca cgtgttgcag ctgagctgga ggatggtcag 60
tacgtcaacc tgggaatcgg aatgccaact ctgattcctg gacacattcc tgctggcaag 120
gatgtgatcc ttcactctga gaacggaatc cttggtgttg gaccataccc aactgatgac 180
gaggttgatc ctgaactgat caatgctggc aaggagacca tcactgttgt tcctggagga 240
gcattcttct cctcatctga ctcattctcc atgatccgtg gtggctctgt cgatgttgct 300
gttcttggtg caatgcaggt ctcatcacat ggtgatctgt ccaactggat ggttcctggt 360
gcaatggtga agggaatggg aggagcaatg gatcttgttc atggagcagg caaggtgatc 420
gtcatgatgg accacgtgac caagaagggt gagcacaaaa tcttggagga gtgcactctg 480
ccatacactg gcaagcgttg cgtgcagaag atcgtgactg atctggctgt gatcgacgtg 540
actcctgatg gactgcgtct ggttgagact gcacctggac actctgttga ggatgttcag 600
gctgcaactg gtgccactct gcttgttgat gccactgtgt aa 642
<210> 16
<211> 1212
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgcctgaag ctgtgatctg cgaaccactt cgtactcctg ttggacgttt cggtggacag 60
ttccgtgaca tctctgcaca agcactggct gctactgtga tcactgaact ggttgcacgt 120
actggcatct ctggtgctga catcgacgac gtcatccttg gacaggcatc acctaacggt 180
gatgcacctg ctatcggacg tatcgctgca ctggacgctg accttggcat cgatgttcct 240
ggtatgcaga tcgaccgtcg ttgtggatct ggactccaag ctgtgctgca agcctgtatg 300
caggttcagt ctggtggcaa cgatctggtt ctggctggtg gtgtggagtc catgtcacag 360
accgagttct atgcaactgg tatgcgttgg ggtgtgaagg ctgaggctgt tgcactgtct 420
gatcgtcttg cacgtgcacg tgtgactgct ggtggcaaga acttccctgt tcctggtggc 480
atgatcgaga ctgctgagaa ccttcgtcag gagttctcca tctcacgtgc tgaccaggat 540
gcacttgctg ttcagtcaca ccgtcgtgct gttgctgcac agaactctgg tgtcttcgct 600
gaagagatcg ttggtgtctc tgttccacag cgtaagtccg agcctgtgct ggtgtcaact 660
gatgagcatc cacgtgctga cactactgtt gagtcactgg ccaagctgaa ggccatccgt 720
gcatccatcg atcctgagtc cactgtcact gctggcaatg catctggaca gaacgatggt 780
gcagcaatcg caatcgtgac tactcctgag aaggctgctg cactgggact tcgtccactt 840
gcacgtcttg catcatgggg tgttgctggt gttgcaccac gtactatggg catcggacct 900
gttcctgcat ctgagaaggc acttggacgt cttggactga ctcttgatga catgggtgtg 960
atcgaactga acgaagcatt cgctgcacag gcactggctg tgactcgttc atggggtatc 1020
gagtctgatg atccacgtct gaacccacat ggttctggca tctcacttgg acatcctgtt 1080
ggtgctactg gtgcacgtat ccttgccact ctgcttcgtg agatggatcg tcgtgaagtt 1140
cgttacggac tggagaccat gtgcattggt ggtggacagg gacttgctgc tgtcttcgaa 1200
cgtctgtcat aa 1212
Claims (7)
1.一种可完全降解多环芳烃植物的培育方法,包括以下步骤:
1)按照植物表达模式,对假单胞菌来源的多环芳烃降解基因簇进行优化重构,构建成包含十价多环芳烃降解基因的由植物启动子控制的多环芳烃降解表达载体,能使多环芳烃降解到邻苯二酚;所述多环芳烃降解基因簇来自恶臭假单胞菌的NahAa、NahAb、NahAc、 NahAd、NahB、NahC、NahD、NahE、NahF和NahG基因;优化重构后,十价多环芳烃降解基因的具体核苷酸序列依次如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10所示;所述多环芳烃为萘;
2)按照植物表达模式,对微生物来源的邻苯二酚降解基因簇进行优化重构,构建成包含六价邻苯二酚降解基因的由植物启动子控制的邻苯二酚降解表达载体,能使邻苯二酚降解到乙酰辅酶A;所述邻苯二酚降解基因簇来自红球菌的CatA、CatB、CatCD、CatI、CatJ和CatF基因;优化重构后,六价邻苯二酚降解基因的具体核苷酸序列依次如SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15和SEQ ID NO.16所示;
3)将多环芳烃降解表达载体和邻苯二酚降解表达载体通过农杆菌介导共转化到同一植物中。
2.根据权利要求1所述可完全降解多环芳烃植物的培育方法,其特征在于,步骤1)及步骤2)中,按照植物偏爱密码、RNA结构稳定性及蛋白翻译效率对多环芳烃降解基因簇和邻苯二酚降解基因簇进行优化重构。
3.根据权利要求1所述可完全降解多环芳烃植物的培育方法,其特征在于,步骤3)中,将构建的多环芳烃降解表达载体和邻苯二酚降解表达载体通过电击法导入根癌农杆菌中,所述根癌农杆菌为为EHA105、LBA4404或GV3101。
4.根据权利要求1所述可完全降解多环芳烃植物的培育方法,其特征在于,步骤3)中,所述植物为拟南芥或水稻,转化后利用潮霉素和草胺磷筛选双抗筛选标记植物。
5.一种用于植物的多环芳烃降解表达载体,其包括10个不同的多环芳烃降解基因表达单元,各多环芳烃降解基因表达单元通过同尾连接酶方法全部连接在植物表达载体上;其中,所述多环芳烃降解基因分别为来自恶臭假单胞菌的NahAa、NahAb、NahAc、NahAd、NahB、 NahC、NahD、NahE、NahF和NahG基因按植物表达模式优化后的基因,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点;所述多环芳烃降解基因表达单元由一个按植物表达模式优化后的多环芳烃降解基因、组成型启动子CaMV35SΩ启动子和NOS终止子组成,所述多环芳烃降解基因的具体核苷酸序列如SEQ ID NO.1、SEQ IDNO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示。
6.一种用于植物的邻苯二酚降解表达载体,其包括6个不同的邻苯二酚降解基因表达单元,各邻苯二酚降解基因表达单元通过同尾连接酶方法全部连接在植物表达载体上;其中,所述邻苯二酚降解基因分别为来自红球菌CatA、CatB、CatCD、CatI、CatJ和CatF基因按植物表达模式优化后的基因,每个表达单元的5'域添加有EcoRI和XhoI酶切位点,3'域添加有SalI和HindIII酶切位点;所述邻苯二酚降解基因表达单元由一个按植物表达模式优化后的邻苯二酚降解基因、组成型启动子CaMV35SΩ启动子和NOS终止子组成,所述邻苯二酚降解基因的具体核苷酸序列如SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ IDNO.14、SEQ ID NO.15或SEQ ID NO.16所示。
7.如权利要求5或6所述的降解表达载体,其特征在于,所述多环芳烃降解表达载体或邻苯二酚降解表达载体中删除了原始表达载体中报告基因的表达盒,并用EcoRI、SalI、BamHI、KpnI、XbaI和HindIII替换了原始表达载体中的多克隆位点片段。
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