Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston) (Pectinoida, Pectinidae)

Zhikong scallop Chlamys farreri(Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

Domain analysis of the Edwardsiella tarda ferric uptake regulator

Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75074, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (I) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of Fur(Et).

The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein

MEP is a member of thioester-containing protein (TEP) family found in Zhikong scallop Chlamys farreri and is involved in innate immunity against invading microbes. In the present study, the genomic DNA of CfTEP was cloned and characterized. The genomic DNA sequence of CfTEP consisted of 40 exons and 39 introns spanning 35 kb with all exon-intron junction sequences agreeing with the GT/AG consensus. The genomic organization of CfTEP was similar to human and mouse 0 rather than ciona C3-1 and Drosophila dTEP2. By RT-PCR technique, seven different cDNA variants of CfTEP (designated as CfTEP-A-CfTEP-G) were cloned from scallop gonad. CfTEP-A-CfTEP-F were produced by alternative splicing of six mutually exclusive exons (exons 19-24), respectively, which encoded the highly variable central region. While in CfTEP-G, the deletion of all the six exons introduced a new translation stop site and might trigger nonsense mediated decay (NMD). The mRNA expression and the proportion of the seven CfTEP variant transcripts were examined in the gonad of scallops after bacterial challenge. The fragments containing the highly variable central region of UTEP were amplified by RT-PCR and a 100 positive clones were sequenced randomly. The expression profiles of the seven MEP variants were different and displayed the sex and bacteria dependent manner. In the blank, sea water and Listonella anguillarum challenged subgroups of male scallops, all the transcripts detected were CfTEP-G isoform. In the Micrococcus luteus challenged subgroup, the isoforms expressed and their proportions were CfTEP-F (54%), CfTEP-B (23%), CfTEP-A (10%), CfTEP-C (7%) and CfTEP-E (6%). However, in the gonad of female scallops, only CfTEP-A were found in blank and sea water challenged subgroups. After L anguillarum or M. luteus challenge, four and five isoforms were detected, respectively, with CfTEP-F isoform being the most one in the both subgroups. These results suggested that the evolution of TEP genes was very complex, and that the diverse CfTEP transcripts generated by alternative splicing played an important role as pattern recognition receptors in the innate immune defense of scallops. (C) 2009 Elsevier Ltd. All rights reserved.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

There are two 5 '-flanking regions of bkt encoding beta-carotene ketolase in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a commercially valuable astaxanthin, with levels reaching up to 4% dry weight under environmental stress. In recent years, much effort has been devoted to understanding the molecular mechanisms regulating astaxanthin biosynthetic pathways. Beta-carotene ketolase (bkt), with control being exhibited at the transcription level, plays an important role in astaxanthin biosynthesis by H. pluvialis. Here we demonstrate the presence of two separate 5'-flanking regions [1.5 kilobase (kb) and 2 kb] of bkt (bkt1 and bkt2) that possess regulatory elements similar to those of known stress-responsive genes in plants. Results of 5'-deletion constructs and transient beta-galactosidase expression assays demonstrate that there may be positive regulatory elements governing expression in the shorter promoter at -1060/-900 from the 1.5 kb 5' region, and in the longer promoter at -1838/-1219 and at -1046/ -734 from the 2 kb 5' region relative to each homologous ATG start codon. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5 kb 5' untranslated region of bkt1 may function as a negative regulatory element to regulate its own promoter.

鳗弧菌rpoS功能的研究

rpoS 基因编码RpoS因子（RNA聚合酶的σ亚基），能增强细菌细胞对外界不良环境的抗逆性和适应能力。我们从鳗弧菌（Vibrio anguillarum）M3 Fosmid 文库中得到rpoS 基因序列，全长999bp，同源分析发现与大肠杆菌（Escherichia coli）有71%的相似性，与霍乱弧菌（Vibrio cholerae ）有78%的相似性。为了研究rpoS在鳗弧菌中的作用，我们利用in-frame deletion技术构建了rpoS 基因的非极性缺失突变株，同时利用细菌的接合转移，以低拷贝克隆质粒pSUP202为载体，构建了突变株的互补株。 在TSB培养基中，rpoS 基因的缺失减缓了鳗弧菌对数期的生长，但是对稳定期的生长没有影响。对数期鳗弧菌在1M蔗糖（渗透压胁迫）和5%（v/v）乙醇中的生长减缓，而在18%(v/v)乙醇中的生长及42℃热击时的存活率相对于野生型没有变化。不同生长时期的鳗弧菌对15mM H2O2的反应有所不同，rpoS 的缺失使对数期的鳗弧菌对15mM H2O2的反应更加敏感。在rpoS基因互补株中，上述表型几乎恢复到野生型水平。 我们通过感染实验发现，rpoS 基因的缺失使鳗弧菌的LD50提高了20倍。 RpoS的突变对鳗弧菌的泳动和胞外酶的产生也有影响。突变株泳动圈直径是野生型的73.8%，在明胶平板和酪蛋白平板上的透明圈直径分别为野生型的61%和69%。通过azocaseion检测胞外产物ECP酶活发现，突变株的酶活是野生型的35.5%。 以上数据表明了RpoS在鳗弧菌对数期应对外界不良环境时起到了重要作用，同时参与了鳗弧菌的致病过程。

迟缓爱德华氏菌基因组fosmid文库的构建、opp基因簇的克隆及aroA基因缺失突变株的构建

本研究构建了迟缓爱德华氏菌(Edwardsiella tarda)LSE40 基因组fosmid文库，该文库共包含2 500个克隆，插入片段平均大小为33.6kb，文库总容量约84Mb，覆盖E.tarda LSE40基因组（按5Mb计算）超过16倍。随机挑取1 000个fosmid克隆进行双末端测序共得到1 741条高质量的序列，序列平均长度546 bp，全长949 997bp，约为E.tarda基因组的19％。将这些序列提交到KEGG自动注释服务器KAAS对所得序列进行代谢途径分析，得到E.tarda LSE40的KO (KEGG Orthology) 注释。分析结果表明，与代谢途径相关的基因有932条序列，与环境信息处理相关基因283条，与遗传信息处理相关基因220条，与细胞进程和人类疾病相关的基因分别为64条和16条。同时将序列进行BlastX，按照微生物致病性共同主题找到61个毒力相关基因。Fosmid文库的建立和部分基因组序列的生物信息学分析为进一步研究E.tarda LSE40的致病机制、代谢机制和生理生态机制提供了丰富的物质基础。 通过比较基因组的方法，从E.tarda LSE40 fosmid文库克隆到编码寡肽透过酶的opp基因簇，该基因簇全长6 741bp，含有5个ORF，依次编码OppA-B-C-D-F 5个蛋白；位于oppA和oppB的间隔区和oppF之后的非编码区各有一个茎环结构，推测分别为oppA和opp基因簇的转录终止子。以细菌OppA的保守结构域SBP_bac_5构建系统发生树，结果显示E.tarda LSE40与同属细菌E.ictaluri的亲缘关系最近，与肠杆菌科细菌的亲缘关系较近，与革兰氏阳性细菌的亲缘关系较远，表明OppA的SBP_bac_5结构域可作为细菌分类鉴定的依据。 从E.tarda LSE40 fosmid文库克隆aroA基因全序列，该序列全长1 287bp，编码428个氨基酸，与鲶鱼爱德华氏菌(E. ictaluri)氨基酸相似性在94%，与其他肠杆菌科菌如Escherichia coli和Yersinia enterocolitica相似性在73%-74%。通过In-frame deletion构建了E.tarda LSE40 aroA缺失突变株。与野生型相比，aroA突变株的半数致死量LD50提高了62倍。在牙鲆接种~106cfu/ml的E.tarda细菌时，接种野生型细菌的牙鲆在6天内全部死亡，濒死鱼的细菌数达7.97×108cfu/ 100mg；而接种aroA突变株的牙鲆没有出现死亡，28天后检测不到细菌的存在。实验结果为进一步评价aroA突变株作为减毒活疫苗打下了基础。

空值环境下关系数据库的更新 Ⅱ：更新处理的插入、删除和修改操作算法

本文的这部分是以扩展模型为基础，以五种运算为工具，对空值环境下关系数据库的更新处理策略做了深入研究，并给出了相应的算法及算法分析。

A型行为核心特征与血管紧张素转换酶基因型相关性研究

In order to study the role of inherited factors and Type A Behavior Pattern (TABP) in the development of CHD, the present study chose the angiotensin I-converting enzyme (ACE) gene as the target gene, and investigated the associations of TABP, the polymorphism of ACE gene with susceptibility to development of CHD in the healthy population and CHD patients from Northern China. 1. Correlation Analysis Between TABP and serum level of ACE in Chinese healthy individuals TABP and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and the serum level of ACE. 2 The distribution charicteristics of ACE gene polymorphism frequencies and association with serum level of ACE in Chinese healthy individuals population The polymorphism of ACE gene and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that: the ethnic differences in I/D polymorphism of ACE gene are obvious; deletion polymorphism of the ACE gene is associated with serum ACE level. 3. The relationship between insertion/deletion polymorphism of ACE gene and CHD in a Chinese population I/D polymorphism in intron 16 of the ACE gene was determined by polymerase chain reaction(PCR) in a study of 109 patients with CHD. The results showed: The frequencies of DD genotype(0.39) and D allele(0.63) were higher among the CHD group than among the control subjects(0.12 and 0.42 respectively, P < 0.01). Furthermore, MI and multivessel disease was more strongly associated with (P < 0.01). It is indicated that D allele and DD gentype of ACE might be an important risk factor for CHD, especially for MI or multivessel disease in Chinese population. 4. Correlation Analysis Between Type A Behavior Pattern and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 291 Chinese healthy individuals. The result showed that the higher frequency of rare D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene was found in type A behavior individuals compared with type B behavior individuals in 291 healthy individuals; there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. It is suggested that the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience may be associated with heredity. 5. Correlation Analysis Between Angiotensin I-Converting Enzyme Gene Polymorphism, Type A Behavior Pattern and Coronary Heart Disease in Chinese The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 109 patients with CHD. The results showed the development of coronary heart disease(CHD) is influenced mainly by the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience; the deletion polymorphism of ACE gene may be play a important role in the process of it. 6. Correlation Analysis Between Type A Behavior Pattern Core Components and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in1306 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. Furthermore, the behavioral attributes of hostility, being irritated easily and impatience may be associated with heredity. At the end of this research, in terms of theory, the research approaches of TABP and the factors influenced the relationship between TABP and CHD were explored and discussed. Furthermore, several new opinions were put forward.