基因检测英语论文

基因检测英语论文

Far-reaching significance of genetic testing: All diseases are genetically related. Genes are DNA molecules carry the genetic information of the function of fragments of biological material to pass genetic information. Genes for life itself, is the root of life, sickness and death. 1, predictive medicine. In health and sub-health era will be able to accurately predict the risk of disease. 2, disease prevention. Disease = internal + external factors. Through the understanding of internal factors, which can effectively avoid the impact of external factors, thereby reducing the risk of illness. 3, health management. Through genetic testing, knowing that they have certain aspects of disease susceptibility genes, we can take the initiative for improving the environment and living habits,
Make their own health management. 4, personalized health care services. Through genetic testing, prompt us to what medicines to be used with caution, not only significantly reduce unnecessary medical expenses, increased efficacy, but also to avoid causing more harm to the human body.基因检测深远的意义： 一切疾病都与基因有关。基因是 DNA 分子上携带有遗传信息的功能片断，是生物传递遗传信息的物质。基因主宰生命，是生命生老病死的根源。 1 、预测医学。在健康和亚健康时期就能够准确预测疾病的风险。 2 、疾病预防。疾病＝内因＋外因。通过对内因的了解，可有效地避免外因的影响，从而可降低患病的风险。 3 、健康管理。通过基因检测，知道自己有某方面的疾病易感基因，就可主动的改善环境和生活习惯，做好自己的健康管理。 4 、个性化医疗服务。通过基因检测，提示我们哪些药物要慎用，不但大大地降低了不必要的医疗支出，提高了疗效，更避免了对人体造成更大的伤害。

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Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate
by Escherichia coli transformant cells coexpressing
the carbonyl reductase and glucose dehydrogenase genes
由共表达碳酰还原酶和葡萄糖脱氢酶的大肠杆菌转化细胞合成
纯光学（S）-4-氯-3-羟基丁酸乙酯

Abstract The asymmetric reduction of ethyl 4-chloro-3-
oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate
((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the
catalyst. In an organic-solvent-water two-phase system,(S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.
本本篇文献研究了利用COBE不对称合成（S）-4-氯-3-羟基丁酸乙酯（CHBE）。大肠杆菌细胞作为催化剂同时表达了来自念珠菌属magnoliae的碳酰还原酶和来自巨大芽孢杆菌的葡萄糖脱氢酶基因。在水/有机溶剂两相体系中，(S)-CHBE在有机相中的浓度可以达到2.58M（430g/l），摩尔产率达到85%。大肠杆菌的副产物S1和GDH也达到了1.25M（208g/l），COBE在水相中不稳定，所以(S)-CHBE可以在水单相中不停的生成。在这种情况下，适当的从NADP+到CHBE的转变达到了21,600 mol/mol。所形成的CHBE的旋光度在这种体系中100%对映体过量。在水相中用携带含有S1和GDH基因质粒的E. coli HB101作为催化剂不对称还原是比较简单的。并且，这种体系并不额外需要商业GDH或者有机溶剂。因此，这种体系对于实际合成纯光学活性的(S)-CHBE是非常方便的。

Optically active 4-chloro-3-hydroxybutanoic acid esters are useful chiral building blocks for the synthesis of pharmaceuticals. The (R)-enantiomer is a precursor of L-carnitine (Zhou et al. 1983), and (S)-enantiomer is an important starting material for hydroxymethylglutaryl- CoA (HMG-CoA) reductase inhibitors (Karanewsky et
al. 1990). Many studies have described the microbial or enzymatic asymmetric reduction of 4-chloro-3-oxobutanoic acid esters (Aragozzini and Valenti 1992; Bare et al.1991; Hallinan et al. 1995; Patel et al. 1992; Shimizu et al. 1990; Wong et al. 1985) based on the reduction by baker’s yeast (Zhou et al. 1983).We have previously showed that Candida magnoliae AKU4643 cells reduced ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96% enantiomeric excess (e.e.) (Yasohara et al. 1999). As this yeast has at least three different stereoselective reductases (Wada et al. 1998, 1999a, b), the (S)-CHBE produced by this yeast was not optically pure. From among these three enzymes, an NADPH-dependent carbonyl reductase, designated as S1, was purified and characterized in some detail (Wada et al. 1998). We cloned and sequenced the gene encoding S1 and overexpressed it in Escherichia coli cells. This E. coli transformant reduced COBE to optically pure (S)-CHBE in the presence of glucose, NADP+, and commercially available glucose dehydrogenase (GDH) as a cofactor generator (Yasohara
et al. 2000).
Here, we describe the construction of three E. coli transformants coexpressing the S1 from C. magnoliae and GDH from Bacillus megaterium genes and analyze the reduction of COBE catalyzed by these strains. Previous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical purity of 92% e.e. (Kataoka et al. 1999; Shimizu et al. 1990) recommended an organic- solvent two-phase system reaction for an enzymatic or microbial reduction, because the substrate (COBE) is unstable in an aqueous solvent and inactivates enzymes. We examined the reduction of COBE to optically pure (S)-CHBE by E. coli transformants in a water monophase system reaction and discuss the possible use of this type of reaction system in industrial applications。

具有旋光性的（S）-4-氯-3-羟基丁酸乙酯在药物制剂的合成中是重要的手性化合物。其右旋体是L-卡尼汀的前体，其左旋体是羟甲基戊二酰辅酶A还原酶抑制剂的起始材料。许多研究描述了以面包酵母为基础微生物或者酶的COBE的不对称还原。我们先前已经知道利用来自念珠菌属magnoliae AKU4643 细胞催化COBE生成光学纯度96%的CHBE。这种酵母至少有三种立体选择性的还原酶，这种酵母产生的CHBE并非纯光学的，在这三种酶之中，NADPH-依赖碳酰还原酶，我们克隆并测序编码S1的基因，并在大肠杆菌中过表达。大肠杆菌转化细胞在葡萄糖，NADP+和商业化的葡萄糖脱氢酶作为辅酶因子的启动子催化COBE生成纯光学的CHBE。
我们构建这三种大肠杆菌转化细胞共表达来自的S1和来自巨大芽孢杆菌的GDH，并分析COBE被这几种菌株催化还原的反应机理。先前的报道表明，利用酶催化还原COBE生成CHBE光学纯度可达92%，也提到了因为底物（COBE）在水相中不稳定，并且酶容易钝化，所以利用酶或者微生物在有机溶剂/水两相体系中催化反应。我们研究了在水单相体系中由COBE还原生成纯光学的CHBE，还讨论了这种反应体系在工业应用中可能的用途。

Materials and methods
Bacterial strain and plasmids
The E. coli strains used in this study were JM109 and d pGDA2, in which the GDH gene from B. megaterium is inserted into pKK223-3, was kindly provided by Professor I. Urabe, Osaka University (Makino et al. 1989). Plasmids pSL301 and pTrc99A were purchased from Invitrogen (USA), and Amersham Pharmacia Biotech (UK), respectively. Plasmids pUC19 and pSTV28 (Homma et al. 1995; Takahashi et al. 1995) were purchased from Takara Shuzo (Japan).
材料和方法
菌株和质粒
本次实验中使用的大肠杆菌是JM109 and HB101。来自B. megaterium的GDH基因插入到Pkk233-3质粒中，而带有GDH基因片段的pGDA2质粒由到由大阪大学的urabe教授提供。质粒pSL301和 pTrc99A是由美国的Invitrogen公司和英国的公司分别购买的。质粒pUC19和pST28是由日本takara公司购买的。
The recombinant plasmid used in this study was constructed as follows (Fig. 1): Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kilobase pairs (kb) including the GDH gene. This fragment was inserted into the EcoRI-PstI site of plasmid pSL301 to construct plasmid pSLG. Plasmid pSLG was double-digested with EcoRI and XhoI to isolate a DNA fragment of about 0.9 kb including the GDH gene.
这次实验使用的重组质粒构建如下：质粒pGDA2 被EcoRI 和 PstI双酶切从而分离出一个大小约为0.9kb的包含有GDH基因的DNA片段。这个片段被插入到质粒Psl301的EcoRI-PstI酶切位点从而构建出质粒pSLG。质粒pSLG被EcoRI和XhoI

To construct plasmid pNTS1G, this 0.9-kb fragment was inserted into the EcoRI-SalI site of pNTS1, which was constructed to overproduce S1 as described previously (Yasohara et al. 2000). To construct plasmid pNTGS1, plasmid pNTG was first generated. Two synthetic primers (primer 1, TAGTCCATATGTATAAAGATTTAG,and primer 2 TCTGAGAATTCTTATCCGCGTCCT) were prepared for polymerase chain reaction (PCR) using pGDA2 as the template. The PCR-generated fragment was double- digested with NdeI and EcoRI and then inserted into the NdeI EcoRI site of plasmid pUCNT, which was constructed from pUC19 and pTrc99A, as reported (Nanba et al. 1999), to obtain pNTG. To construct plasmid pNTGS1, two synthetic primers (primer 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and primer 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC) were prepared using pUCHE, which contains the S1 gene as the template. The PCR-generated fragment was double-digested with EcoRI and SalI and then inserted into the EcoRI-SalI site of pNTG to obtain pNTGS1. Plasmid pNTS1G, pNTGS1 or pNTG was transformed into E. coli HB101.
构建pNTS1是为了过表达前文所提到的S1，这个0.9kb大小的片段被插入到pNTS1的EcoRI-SalI酶切位点从而构建pNTS1G。为了构建质粒pNTGS1，首先需要构建pNTG。两个合成引物（引物1，TAGTCCATATGTATAAAGATTTAG和引物2，TCTGAGAATTCTTATCCGCGTCCT）和作为模板的pGDA2是PCR反应需要的。PCR得到的片段是由NdeI 和EcoRI双酶切和并插入到质粒pUCNT的NdeI EcoRI酶切位点来得到pNTG。根据报道，pUCNT是由pUC19和 pTrc99A构建而来。为了构建质粒pNTGS1，两个合成引物(引物 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and 引物 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC)，包括了S1基因作为模板。Pcr产物片段被EcoRI和SalI双酶切然后被插入到pntg的EcoRI-SalI酶切位点得到pntg1.质粒pNTS1G, pNTGS1或者 pNTG都是导入大肠杆菌HB101.
Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kb including the GDH gene. To construct plasmid pSTVG, this fragment was inserted into the EcoRI-PstI site of plasmid pSTV28. Plasmid pSTVG was transformed into E. coli HB101.
质粒pGDA2被EcoRI 和 PstI双酶切得到包含GDH基因的0.9kb大小的DNA片段。为了构建pSTVG质粒，这个片段被插入到pSTV28质粒的EcoRI-PstI的酶切位点。pSTVG质粒被导入到E. coli HB101。

Medium and cultivation
The 2×YT medium comprised 1.6% Bacto-tryptone, 1.0% yeast
extract, and 0.5% NaCl, pH 7.0. E. coli HB 101 carrying pNTS1,
pNTG, pNTS1G, or pNTGS1 was inoculated into a test tube containing
2 ml 2×YT medium supplemented with 0.1 mg/ml ampicillin,
followed by incubation at 37 °C for 15 h with reciprocal shaking.
This preculture (0.5 ml) was transferred to a 500-ml shaking
flask containing 100 ml 2×YT medium. The cells were cultivated
at 37 °C for 13 h with reciprocal shaking. E. coli HB101 carrying
pNTS1 and pSTVG was similarly cultivated in 2×YT medium
supplemented with 0.1 mg/ml ampicillin and 0.1 mg/ml chloramphenicol.
培养基和培菌
2*YT培养基 包含有1.6%细菌用胰蛋白胨，1.0%酵母提取物，0.5% NaCl，pH7.0.
携带有pNTS1,pNTG, pNTS1G, 或 pNTGS1的大肠杆菌HB101被接种到有0.1mg/ml氨苄青霉素的2ml的2*YT培养基，37°C摇床15小时。将0.5ml菌液接种到100ml2*YT培养基的500ml烧瓶中。在37°C摇床培养13小时。携带有pNTS1 和 pSTVG质粒的大肠杆菌HB101在2*YT培养基中培养方法相似，只是培养基中要加入0.1 mg/ml的氨苄青霉素和 0.1 mg/ml的氯霉素。

Preparation of cell-free extracts and the enzyme assay
Cells were harvested from 100 ml of culture broth by centrifugation, suspended in 50 ml of 100 mM potassium phosphate buffer (pH 6.5), and then disrupted by ultrasonication. The cell debris was removed by centrifugation; the supernatant was recovered as the cell-free extract. Carbonyl reductase S1 activity (COBE-reducing activity) was determined spectrophotometically as follows: The assay mixture consisted of 100 mM potassium phosphate buffer (pH 6.5), 0.1 mM NADPH, and 1 mM COBE. The reactions were incubated at 30 °C and monitored for the decrease in absorbance at 340 nm. The assay mixture for GDH activity consisted of 1 M Tris-HCl buffer (pH 8.0), 100 mM glucose, and 2 mM NADP+. The reactions were incubated at 25 °C and monitored for the increase in absorbance at 340 nm. One unit of S1 or GDH was defined as the amount catalyzing the reduction of 1 μmol NADP+ or oxidation of 1 μmol NADPH per minute, respectively. Protein concentrations were measured with a protein
assay kit containing Coomassie brilliant blue (Nacalai Tesque, Japan),
using bovine serum albumin as the standard (Bradford 1976).
无细胞抽提液和酶鉴定
将100ml培养液离心收获菌体，用50ml0.1mol/LpH为6.5的磷酸缓冲液悬浮，然后超声粉碎。细胞碎片通过离心可以去除，收集上层清液就是无细胞抽提物。碳酰还原酶S1的活性由分光光度计测量如下：测定的混合物包括：0.1mol/LpH6.5的磷酸二氢钾缓冲液，0.1mMNADPH和1mMCOBE。反应在30°C条件下反应，并且随时监测其在340nm处的吸光值。测GDH混合物包括：1M pH 8.0的Tris-HCl的缓冲液，100mM的葡萄糖，2mM的NADP+。反应在25°C下进行，监测其在340nm处的吸光值。一个单位S1或GDH被定义为每分钟催化还原1μmol NADP+或氧化1 μmol NADPH的量。蛋白质的测定通过含有考马斯亮蓝的蛋白质测定试剂利用牛血清白蛋白作为标准进行测定。

Study of enzyme stability
One milliliter of 100 mM potassium phosphate buffer (pH 6.5) containing the cell-free extracts of E. coli HB101 carrying pNTS1 (S1: 20 U/ml) was mixed with an equal volume of each test organic solvent in a closed vessel. After the mixture was shaken at 30 °C for 48 h, the remaining enzyme activities in an aqueous phase were assayed as described above. The mixture, containing 100 mM potassium phosphate buffer (pH 6.5), S1 (20 U/ml), and various concentrations of CHBE, was incubated at 30 °C for 24 h in order to study the enzyme’s stability in the presence of remaining enzyme activities were assayed as described above.
酶稳定性的研究
一毫升含有含有pNTS1质粒的E. coli HB101的无细胞抽提液的100mM磷酸氢二钾缓冲液（pH6.5）与等体积的有机溶剂混合。混合物在30 °C震摇48小时后，水相中残留的酶活力即是上述的酶活力。
COBE reduction with E. coli cells expressing the S1 gene and E. coli cells expressing GDH genes in a two-phase system reaction
The reaction mixture comprised 15 ml culture broth of E. coli HB101 carrying pNTG, 17 ml culture broth of E. coli HB101 carrying pNTS1, 1.6 mg NADP+, 4 g glucose, 2.5 g COBE, 25 ml n-butyl acetate, and about 25 mg Triton X-100. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. At 2 h, 1.25 g COBE and 2.5 g glucose were added to the reaction mixture. To compare the reaction by E. coli transformant coexpressing the GDH and S1 genes, 30 ml culture broth of E. coli
HB101 carrying pNTS1G was used instead of culture broth of E. coli HB101 carrying pNTG and E. coli HB101 carrying pNTS1. Other components and the procedure were the same as described above.
表达S1基因和GDH基因的大肠杆菌细胞在两相反应体系中的还原反应
混合物包含有带有pNTG质粒的大肠杆菌HB101的菌液15ml，pNTS1质粒的大肠杆菌HB101的菌液17ml，1.6 mg NADP+，4 g葡萄糖，2.5g的COBE，25ml的n-butyl acetate丁酰醋酸盐和大约25mg的聚乙二醇辛基苯基醚Triton X-100。用5M的NaOH溶液将pH控制在6.5。在反应两小时后，加入1.25gCOBE和2.5g葡萄糖到该混合物中。比较大肠杆菌转化细胞共表达GDH和S1基因，携带有pNTS1G质粒的大肠杆菌HB10130ml菌液取代了携带有pNTG和pNTS1质粒的大肠杆菌HB101菌液。其他的成分和步骤和上述的方法相似。
COBE reduction to (S)-CHBE in a two-phase system reaction
The reaction mixture contained 50 ml of culture broth of an E. coli HB101 transformant, 3.2 mg NADP+, 11 g glucose, 10 g COBE, 50 ml n-butyl acetate, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C, and the pH was controlled at 6.5 with 5 M sodium hydroxide. Five grams of COBE/5.5 g glucose and 10 g COBE/11 g glucose were added to the reaction mixture at 3 h and 7 h, respectively; 3.2 mg NADP+ was added at 26 h.
COBE在两相系统中还原生成（S）-CHBE
反应混合物包含50ml E. coli HB101转化细胞的培养液，3.2mgNADP+,11g
葡萄糖，10gCOBE，50ml丁酰醋酸，和大概50mg聚乙二醇辛基苯基醚Triton X-100.
在30°C温度下将其混合均匀，并用5M的NaOH溶液将pH控制在6.5。在第3小时加入5gCOBE和5.5g葡萄糖或者在第7小时加入10gCOBE和11g葡萄糖，分别在第26小时加入3.2gNADP+。
COBE reduction to (S)-CHBE in an aqueous system reaction
The reaction mixture was made up of 50 ml of culture broth of an E. coli HB101 transformant, 3.1 mg NADP+, 11 g glucose, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C. Fifteen grams of COBE was fed continuously by means of a micro-feeding machine at a rate of about 0.02 g/min for about 12 h. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. The reaction mixture was extracted with 100 ml ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then evaporated in vacuo.
COBE在水相中还原成(S)-CHBE的反应
反应的体系是由50ml大肠杆菌HB101转化细胞的菌液，3.1mgNADP+，11g葡萄糖和大约50mg聚乙二醇辛基苯基醚Triton X-100。反应混合物在30°C15mg的COBE通过微量添加机器以0.02 g/min的速率连续12小时恒定的加入到体系中。用5M的NaOH溶液将pH控制在6.5。反应混合物用100ml乙酸乙酯萃取。有机层用无水硫酸钠吸干，并在真空中脱水。
Analysis
The organic layer was obtained on centrifugation of the reaction mixture and was assayed for CHBE and COBE by gas chromatography. Optical purity of CHBE was analyzed by high-performance liquid chromatography (HPLC), as described previously (Yasohara et al. 1999).

Enzymes and chemicals
Restriction enzymes and DNA polymerase were purchased from
Takara Shuzo (Japan). COBE (molecular weight: 164.59) was purchased
from Tokyo Kasei Kogyo (Japan). Racemic CHBE (molecular
weight: 166.60) was synthesized by reduction of COBE with
NaBH4. All other chemicals used were of analytical grade and
commercially available.

分析
离心反应混合物得到的有机层通过气相色谱法测定其CHBE和COBE。COBE的光学纯度如前所述通过高效液相色谱法进行分析。

酶和化学试剂
限制性内切酶和DNA聚合酶由takara公司购得，COBE（分子量：164.59）由东京Tokyo Kasei Kogyo公司购得，消旋体CHBE（分子量166.6）通过COBE及NaBH4合成。所有其他化学试剂都是分析等级和商业化的试剂。

Construction of E. coli transformants overproducing S1 and GDH

To express the carbonyl reductase S1 and GDH genes in the same E. coli cells, four expression vectors were constructed (Fig. 1). Plasmids pNTS1G and pNTGS1 contain the S1 gene from C. magnoliae, the GDH gene from B. megaterium, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. Plasmid pNTS1 contains the S1 gene, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. The enzyme activities in cell-free extracts of the E. coli transformants are shown in Table 1. E. coli HB101 cells carrying the vector plasmid pUCNT had no detectable S1 or GDH activity. E. coli HB101 carrying either pNTS1G or pNTGS1 showed S1 and GDH activity without isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The S1 activities of these two transformants were lower than the GDH activities. To obtain a transformant whose S1 activity was equal to or greater than the level of GDH activity, we used a lower copy vector, pSTV28 (Homma et al. 1995; Takahashi et al. 1995), to express the GDH gene. It may be possible to raise the S1 activity by lowering the GDH activity. Plasmid pSTVG contains the GDH gene, the lac promoter, the chloramphenicol resistance gene, and the replicative origin derived from pACYC184 for compatibility with the plasmid pNTS1. In E. coli HB101 carrying pNTS1 and pSTVG, the S1 activity was higher than the GDH activity, but this GDH
level may be too low to regenerate in a COBE reduction reaction as described below.

过产生S1和GDH的大肠杆菌转化细胞的构建
为了在同一大肠杆菌细胞中表达碳酰还原酶S1和GDH基因，要构建四个表达型载体。质粒pNTS1G 和 pNTGS1包含有来自C. magnoliae的S1基因，来自B. megaterium的GDH基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子，质粒pNTS1包含有S1基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子。在大肠杆菌转化细胞的无细胞抽提物的酶活力如表一所示。携带有运输质粒pUCNT的大肠杆菌细胞无法检测到其S1和GDH活性。携带有pNTS1G 或 pNTGS1质粒在没有IPTG的诱导下有S1和GDH的活性。在这两个转化菌种中，S1的活力小于GDH的活力。为了得到S1活性等于或者大于GDH的大肠杆菌转化菌株，我们使用低拷贝的载体pSTV28，来表达GDH基因。它可能可以通过降低GDH的活性从而提高S1的活性。质粒pSTVG包含有GDH基因，lac启动子，和氯霉素抗性基因，以及与pNTS1具有相容性的从pACYC184得来的复制起始位点。在携带有pNTS1和pSTVG的大肠杆菌转化细胞中，S1的活性要高于GDH的活性，但是GDH的活性可能会太低而在COBE还原反应中不能再生。

太长了，字数有限制，所以不能发完。分数我无所谓啦，我很少登录的。这应该算是基因工程的吧，是我以前自己翻的，不是很好。如果你要的话可以联系我的邮箱。

基因检测用英语怎么说

基因检测英语是Genetic Test。

所谓基因检测，通常是指我们取检测者的血液、口腔粘膜细胞，经提取和扩增其基因信息后，通过基因芯片技术或超高通量SNP分型技术，对被检测者细胞中的DNA分子的基因信息进行检测，分析他所含有的各种疾病易感基因的情况，从而使人们能及时了解自己的基因信息，预测身体患病的风险，进而有针对性地主动改善自己的生活环境和生活习惯，预防和避免重大疾病的发生。简单来说，基因检测报告就如同一本个人健康说明书，它会告诉人们潜在的疾病风险。

想要了解更多有关基因检测的相关信息，推荐咨询海普洛斯。海普洛斯致力于ctDNA液体活检技术的开发与应用，自主研发了高效实用的CUBE-ctDNA单分子编码技术，去除测序错误，结合超高深度高通量测序技术（20,000×，灵敏度达0.05%，特异性大于90%）、自主研发的癌症基因捕获技术HAPCap（High Affinity Probe Capture），覆盖率达99.7%，全面覆盖肺癌、乳腺癌、结直肠癌、胃癌等癌症相关基因、融合基因等，专业值得信赖。【● 没病有必要做基因检测吗？过来人有话说......】

谁能解决一篇遗传育种英汉对译论文？

利用生物技术向小麦导入冰草优异基因的研究

摘 要

小麦是世界上种植面积最大和最重要的粮食作物。利用生物技术向栽培作物转移向外源优异基因来拓宽小麦育种的遗传基础，是现代作物遗传育种学科中的一个非常重要的研究领域。

栽培小麦(Triticum aestivum L., 2n=4x-42, AABBDD)与冰草属(Agropyron Gaertn., P genome )(这儿所说的冰草属是现代小麦族植物分类学上的概念，而非传统的广义冰草属概念，即与一些小麦遗传育种学家将长穗偃麦草(Elytrigia elongata)和中间偃麦草(Elytrigia intermedia)等偃麦草属种也称为冰草的传统概念截然不同)植物间的杂交，可追溯到本世纪30年代(White, 1940; Smith, 1942; Dewey , 1984), 但直到90年代一些学者才先后报道了小麦与冰草属植物间的成功杂交(李立会等， 1990， 1995；Li & Dong, 1991; Chen et al., 1990; Limin & Fowler, 1990; Ahmad & Comeau , 1992; Jauhar, 1992)。 尽管这儿所列出的国外一些科学家也曾获得了小麦与冰草属植物间的杂种，但由于外源种选择的盲然性，即对要从冰草属植物向小麦转移哪些基因不明确(Chen et al., 1990; Jauhar, 1992), 或杂种F1的高度不育性(Ahmad & Comear, 1992), 或要转移的目标基因难以在小麦背景下表达(Limin & Fowler, 1990 )等原因，未见进一步的报道，而是基本上放弃了该领域的研究工作(私人通信，1997)。

在本项研究工作中，我们以普通小麦品种Fukuho (春性，具3对可杂交性基因，农艺性状良好，原产于日本)为母本，以分别采自新疆和内蒙古的3份冰草(Agropyron cristatum <L.>Gaertn., 2n=4x=28, PPPP)为父本进行杂交，并对杂种后代进行了研究。主要结果包括：

1、科学选择远缘杂交亲本，为杂交和外源优异基因转移的成功奠定了坚实的基础。在选择外源供体种的过程中，本项研究首先由中国农业科学院作物品种资源研究所和植物保护研究所、澳大利亚Division of Plant Industry, CSIRO, 加拿大Cytogenetics Section, Ottawa Research Station, Agriculture Canada 等单位对上千份小麦野生近缘植物的农艺性状、抗逆性和抗病性进行了联合鉴定，然后根据综合鉴定结果才精选出本项研究所利用的3份冰草－最佳外源供体种。因为这3份冰草不仅具有其它外源种难以比拟的众多优异基因（性状），包括小麦超高产育种所需的合理株型结构(株高小于60cm且穗下茎长度约占株高的2/3、有效分蘖>50、叶片窄短上挺)、大穗多粒（每穗结实在150粒以上）、黑粒且蛋白质含量极高、极强的抗旱和抗寒性、适度的耐盐性、对三种锈病、白粉病和黄矮病免疫、高抗赤霉病等，而且更为重要的是上述优异基因都是当前小麦育种迫切需要的。

2、利用现代远缘杂交方法和幼胚拯救技术，在国际上首次获得了具部分自交可育性的普通小麦与冰草间的杂种，并发现以不同来源的冰草为父本，不仅杂交结实率不同，而且杂种F1的表现型亦不同。这一结果，一方面突破了前人(Dewey, 1984)所认为的“冰草属P染色体组在小麦族中具有独立的遗传地位，与小麦之间不可能杂交”的论断；另一方面杂种F1具部分自交可育性，为实现外源基因的成功转移 奠定了坚实的物质基础。

3、以杂种F1幼穗为外植体，通过诱导愈伤组织产生体细胞无性系变异，首次发现杂种F1在无染色体数量变异情况下，其自交可育性或从无到有(0Õ 0.032%),或显著提高(10倍)。这一发现之所以重要，是因为它向人们展示了通过这一技术有可能在杂种F1就实现外源基因转移的美好前景。

4、通过精细分析，首次阐明了一些遗传学机理：一是杂种F1自交可育性是由于2条P染色体含有控制减数分裂过程中染色体分离的基因，从而能够形成有功能的近等2n或未减数配子；二是另外1条P染色体上具有抑制小麦Ph基因的遗传因子，能够诱发冰草P染色体组和小麦A、B、D染色体组间的染色体相互配对；三是证实了通过染色体间的自发易位可实现小麦－冰草间的基因交流。这些发现，一方面彻底突破了国际权威所认为的“小麦－冰草间不可能进行基因交流”的论断，另一方面为更加有目的、更加高效率地转移冰草优异基因提供了重要的理论指导。

5、利用回交、选择和形态学、细胞学、等位酶以及基因组原位杂交检测等综合技术，首次育成了11个遗传稳定的小麦－冰草异源二体附加系，并提出了有效产生异源二体附加系（列）的可行做法。异源二体附加系的产生，是研究每条P染色体上的基因在小麦背景下的遗传效应及其有效利用的重要工具。

6、首次创造了一批携带冰草优异基因、遗传稳定(2n=42, 21II ,异源易位系或代换系)且能为育种和生产利用的新种质。其普遍的特点是：有效分蘖多(15~82穗/株)；株高70～95cm且穗下茎约占株高的1/2；旗叶上挺；大穗(55～112粒/穗)；籽粒外观白色或黑色、蛋白质含量高(17.1~20.7%); 千粒重>38g; 综合抗病性（白粉病、条锈病、黄矮病和赤霉病)、抗寒性和抗旱性良好，特别是一些新种质具超高产潜力(理论产量高于600 kg/亩)。目前，这批新种质已为我国小麦主产区的9省15家育种单位利用，其中在陕西、山西有5个新种质正参加产量比较试验。

7、对获得的遗传稳定(2n=42, 21II ,异源易位系)的黑色籽粒和对白粉病免疫新种质中的黑色籽粒基因和抗白粉病基因(均来自冰草)进行了初步遗传分析，证明二者皆为显性单基因遗传。

关键词：普通小麦(Triticum aestivum, L., 2n=6x=42, AABBDD); 冰草(Agropyron cristatum <L.> Gaertn., 2n=4x=28, PPPP)；属间杂种；自交可育性；异源二体附加系；遗传分析；新种质

Introduction of Desirable Genes from Agropyron cristatum (L.) Gaertn. to Triticum aestivum L. Using Biotechnology

Doctoral student : Li-Hui Li

Supervisor: Professor Yu-Shen Dong

(Institute of Crop Germplasm Resources, Chinese Academy of Agriculture Sciences, Beijing, 100081)

Abstract

As in most other crops, the genetic variation of cultivated wheat has been greatly eroded under modern agricultural systems. Genetic erosion not only limits the further improvement of yield and quality but also makes wheat increasingly vulnerable to biological and environmental stresses. A large amount of genetic variation exists in the wild relatives of cultivated wheat. The introduction of genetic variation from alien species has been a valuable method for increasing the amount of genetic diversity available to wheat breeders.

In this experiment, intergeneric hybrids of Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) with three accessions of tetraploid A. cristatum (2n=4x=28, PPPP) were synthesized through immature embryo rescued in artificial medium. These hybrids can be used to: (1) transfer the desirable traits from A. cristatum into common wheat; (2) identify the effects of the P genome on self-fertility in intergeneric hybrids; and (3) produce disomic addition lines of wheat-A. cristatum. Through study of the intergeneric hybrids and their derivatives, the following results were obtained:

1. A. cristatum may be one of the best potential alien donors in the Triticeae for wheat improvement. Agropyron Gaertn. is a small genus of no more than ten species, which constitute what is known as the “crested wheatgrass complex” with the P genome, in accordance with the terminology of many modern botanists. A. cristatum (L.) Gaertn. is the type species of this genus. All species of the genus are very valuable; they are cultivated as predominantly pasture-fodder plants, distinguished by their high level of drought and cold tolerance; some species have be successfully used for fixing drifting sands. In addition, A. cristatum has also been found to possess the other desirable traits that are potentially valuable for wheat improvement through evaluation of all Triticeae collections from the northern part of China; these include shorter stem (usually less than 60 cm), more tillers and florets, immunity to wheat diseases such as rusts, powdery mildew, and barley yellow dwarf virus (BYDV) as well as resistance to wheat scab.

2. In the three hybridization combinations, seed set (0.22%~0.63%) and plant development were different. Each one plant obtained from Fukuho with A. cristatum accession No. Z540 and Z602 respective developed poorly. The former died before heading. Although the later produced two spikes, neither selfed nor backcross seed was obtained from these two spikes. The two plants obtained from the Fukuho×A. cristatum Z559 showed vigorous tillering. This result indicated that the three accessions of A. cristatum used in this experiment are different in crossability with the Fukuho. The root-tips of all hybrid seedlings were observed, and revealed that somatic chromosome number of each was 2n=35 as expected.

3. A self-fertile intergeneric hybrids between Triticum aestivum cv Fukuho (2n=6x=42, AABBDD) and tetraploid A. cristatum (2n=4x=28, PPPP) were obtained for the first contrast with the reports that either no BC1 derivatives from wheat-Agropyron hybrids was obtained or BC1 derivatives obtained were very difficult, in the Fukuho×A. cristatum Z559 hybrids, however, they not only had a high seed set (15.1%) of backcrossing with common wheat, but also were partially self-fertile. The mean configurations at meiotic metaphase I of the hybrids were 24.47 I + 4.32 rod Ⅱ + 0.71 ring Ⅱ + 0.14 Ⅲ + 0.01 Ⅳ. Some of bivalents per cell were clearly heteromorphic on the basis of various chromosome size, indicating that these bivalents were heterogenetic pairing. At anaphase I, chromosome separation was mainly the most (16~30 chromosomes) of 35 chromosomes to assemble at one pole, resulting in that the bigger daughter cells receiving most of 35 chromosomes might develop the functional gametes.

4. In order to induce somaclonal variation, the immature inflorescences of the hybrids between Triticum aestivum cv Fukuho and A. cristatum Z559 were cultured. Although the regenerants did not exhibit variation in chromosome number, they did show a higher degree of meiotic instability than the initial hybrids. Especially, the selfed seed set could be increased greatly in the regenerated plants, being from 0.034% to 0.33%. As a result, a total of 61 selfed seeds were obtained. Obtaining of so more selfed seeds from the Fukuho×A. cristatum Z559 is rare in the intergeneric hybrids involving wheat, and makes a substantial foundation for transferring the desirable genes from A. cristatum into common wheat.

5. Using methods such as morphology, cytology, isozymes and genomic in situ hybridization, the selfed and backcross derivatives were analysed. The results showed that all plants with the alien characters carried the genetic materials of the P genome. Meanwhile, a total of 11 disomic alien addtion lines were obtained.

6. After all cytological data obtained from this experiment were summed up and analysed, some conclusions could be obtained. They were: (1) the A. cristatum Z559 used in this experiment carried a genetic system suppressing Ph activity, and this genetic system might be mainly involved one of the P genome; (2) the P genome contained genes controlling chromosomes segregation at meiotic anaphase, and the genes might be mainly involved two of the P chromosomes; and (3) the spontaneous wheat-A. cristatum translocations can occur in the selfed and backcross derivatives.

7. In this experiment, the other very important result is that some new germplasm with the desirable alien genes were obtained. They showed more effective tillerings (15-82 spikes per plant), plant height ranged from 70 to 95 cm, 55-112 grains per spike, a higher content of protein (17.1-20.7%), resistance to wheat diseases such as powdery mildew, stripe rust, BYDV and wheat scab, and tolerance to drought and cold. So far, all new germplasm obtained from this experiment have been utilized by the 15 institutions for wheat breeding in China.

8. The new germplasm with black-grain in color and immunity to powdery mildew were analyzed. Genetic analysis revealed that these two characters were from A. cristatum Z559 and were controlled by a dominant gene respectively.

Key Words: Triticum aestivum L., Agropyron cristatum, Intergeneric hybrids, Self-fertility, Alien addition and translocation lines, Genetic analysis.