RNA interference reveals chloride channel 7 gene helps short-term hypersalinity stress resistance in Hong Kong oyster Crassostrea hongkongensis

The chloride channel 7 gene(CLC 7)of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC 7.The cDNA was 2572 bp in length,with a 5′non-coding region containing 25 bp,a 3′non-coding region containing 327 bp,and an open reading frame of 2298 bp.ChCLC 7 has 96.8%and 92.1%homology with CLC 7 of Crassostrea gigas and Crassostrea virginica,respectively,and it was clustered with CLC 7 of C.gigas and C.virginica.QRT-PCR showed that ChCLC 7 was expressed in all eight tissues,with the highest in adductor muscle and second in gill.The ChCLC 7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend.After RNA interference,the expression of ChCLC 7 and survival rate of oyster under high salinity stress was reduced significantly,and so did the concentration of hemolymph chloride ion in 48-96 h after RNA interference.We believed that ChCLC 7 could play an important role in osmoregulation of C.hongkongensis by regulating Cl^(-)transport.This study provided data for the analysis of molecular mechanism against oyster salinity stress.

Effects of Salinity on Embryonic Development, Survival, and Growth of Crassostrea hongkongensis

This study examined the effects of salinity on embryonic development, survival, and growth of the Hong Kong oyster Crassostrea hongkongensis. The embryos, larvae, and juveniles of C. hongkongensis were held in artificial seawater at three different salinities(low, 15; medium, 23; and high, 30) to determine the optimum hatchery and nursery conditions for mass production of the seeds. Results showed that the percentage production of straight-hinged larvae from fertilized eggs was significantly lower at the high salinity than at the low- and medium-salinities(P < 0.05). The survival rates of larvae and juveniles differed significantly among the three salinity trials, with the highest survival rate observed at the low salinity(P < 0.05). The shell height of larvae was significantly larger at the low salinity than at the high and medium salinities from days 9 to 15(P < 0.05), whereas that of juveniles was significantly larger at the low salinity than at the high and medium salinities on day 70(P < 0.05). These results indicate that the larvae and juveniles of C. hongkongensis are tolerant to a wide range of salinities(15 to 30), but show better growth and survival at relatively low salinities. Thus, it is recommended to use relatively low salinities in hatchery and nursery systems for improved yields of C. hongkongensis.

Cloning and expression analysis of a ubiquitin gene (Ub_(L40)) in the haemocytes of Crassostrea hongkongensis under bacterial challenge

Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5' untranslated region (UTR) of 34 bp, a 3'-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge.

Dioxyxanthones from Polygala hongkongensis and Their Cytotoxicity

Two new dioxyxanthones, polyhongkongenoxanthones A and B（1 and 2） were isolated from the herbs of Polygala hongkongensis, together with six known xanthones. Their structures were elucidated on the basis of chemical and spectroscopic evidence. The isolates were tested for their cytotoxicity against three tumor cell lines（HepG2, GLC-82 and MCF-7, HepG2=human hepatocellular carcinoma cells; GLC-82=human lung carcinoma cells; MCF-7= human breast carcinoma cells） by MTT assay, among which polyhongkongenoxanthone B（2）, 1,7-dihydroxy-2,3- methylenedioxyxanthone（3） and 1,7-dihydroxy-3,4,8-trimethoxyxanthone（6） are potential antitumor candidate due to their significant cytotoxic effects on the three cell lines..

Megastigmane Glycosides from Polygala hongkongensis Hemsl

Two new megastigmane glycosides,7Z-roseoside,and 7Z-trifostigmanoside Ⅰ,as well as,four known compounds,trifostigmanoside Ⅱ,epimedin C,3＇,7-dihydroxy-4＇-methoxyisoflavone-7-D-glucopyranoside,and formenonetin,were isolated from Polygala hongkongensis Hemsl.The structures of the isolated compounds were established on the basis of UV,IR,NMR,and MS spectral data.Megastigmane glycosides（1-3）were isolated from the family Polygalaceae for the first time.

Current Research and Prospect of <i>Dendrobenthamia hongkongensis</i>in China

Dendrobenthamia hongkongensis is an excellent ornamental tree species, which has a wide range in germplasm resources distribution and rich varieties within species in China and has also a great development prospect of the new superior species for city landscape. So it is necessary for us to do much research for the development and utilization of this species. According to the latest existing research data of D. hongkongensis, the research achievements of D. hongkongensis in Germplasm resource distribution, bio-ecological habits, breeding and cultivation techniques and so on have been analyzed and summed up. On the other hand, its Ornamental value has been utilized in the modern landscape. At the same time, Edible and Medicinal value of D. hongkongensis has been discussed in the paper as well as Material value of D. hongkongensis. In addition, the future aspects of physiological and ecological research, domestication and breeding new varieties, resource protection and landscape application of D. hongkongensis have been proposed.

A New Acylated Anthocyanin from the Red Flowers of Camellia hongkongensis and Characterization of Anthocyanins in the Section Camellia Species

Twelve anthocyanins （1-12） were isolated from the red flowers of Camellia hongkongensis Seem. by chromatography using open columns. Their structures were elucidated on the basis of spectroscopic analyses, that is, proton-nuclear magnetic resonance, carbon 13-nuclear magnetic resonance, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, high resolution electrospray ionization mass and ultraviolet visible spectroscopies. Out of these anthocyanins, a novel acylated anthocyanin, cyanidin 3-O-（6-O-（Z）-p-coumaroyl）-β-galactopyranoside （6）, two known acylated anthocyanins, cyanidin 3-O-（6-O-（E）-p-coumaroyl）-β-galactopyranoside （7） and cyanidin 3-O-（6-O-（E）-caffeoyl）-^- galactopyranoside （8）, and three known delphinidin glycosides （10-12） were for the first time isolated from the genus Camellia. Furthermore, pigment components in C. japonica L., C. chekiangoleosa Hu and C. semiserrata Chi were studied. The results indicated that the distribution of anthocyanins was differed among these species. Delphinidin glycoside was only detected in the flowers of C. hongkongensis, which is a special and important species in the section Camellia. Based on the characterization of anthocyanins in the section Camellia species, there is a close relationship among these species, and C. hongkongensis might be an important parent for creating new cultivars with bluish flower color.

Leptotrichia hongkongensis sp.nov.,a novel Leptotrichia species with the oral cavity as its natural reservoir

A straight, non-sporulating, Gram-variable bacillus (HKU24T) was recovered from the blood culture of a patient with metastatic breast carcinoma. After repeated subculturing in BACTEC Plus Anaerobic/F blood culture broth, HKU24T grew on brucella agar as non-hemolytic, pinpoint colonies after 96 h of incubation at 37 °C in an anaerobic environment and aerobic environment with 5% CO2. Growth was enhanced with a streak of Staphylococcus aureus. HKU24T was non-motile and catalase-negative, but positive for alkaline phosphatase, β-glucosidase, and α-glucosidase. It hydrolyzed phenylphosphonate and reduced resazurin. 16S rRNA, groEL, gyrB, recA, and rpoB sequencing showed that HKU24T occupies a distinct phylogenetic position among the Leptotrichia species, being most closely related to Leptotrichia trevisanii. Using HKU24T groEL, gyrB, recA, and rpoB gene-specific primers, fragments of these genes were amplified from one of 20 oral specimens. Based on phenotypic and genotypic characteristics, we propose a new species, Leptotrichia hongkongensis sp. nov., to describe this bacterium.

Tetrapioid induction of Crassostrea hongkongensis and C.sikamea by inhibiting the polar body 1 release in diploid fertilized eggs

The production of an all-triploid population by mating tetrapioid males with diploid females is the best and most fundamental method for the large-scale production of triploid oysters.Obtaining a stable tetrapioid population is essential for guaranteed production in industrialized triploid cultivation.C.hongkongensis and C.sikamea are important oyster breeding species in southern China,and have great economic value.However,there are not any published data on inducing tetrapioid C.hongkongensis or C.sikamea.Therefore,we investigated tetrapioid induction in these two oyster species by inhibiting the PB1 release in diploid fertilized eggs using Cytochalasin B(CB)under 31℃,15%o salinity.The results confirmed that the optimal tetrapioid induction conditions for C.hongkongensis were a CB concentration of 0.50 mg/L with induction starting at 9.0 min after fertilization,and stopping at 21.0 min after fertilization;the induction efficiency index reached 0.123 under these conditions.The optimal tetrapioid induction conditions for C.sikamea were a CB concentration of 0.50 mg/L,with induction starting at 7.5 min after fertilization and stopping at 18 min after fertilization;the induction efficiency index could be as high as 0.281 under these conditions.However,we confirmed that the tetrapioid rate decreased with larval growth,and no tetrapioids were detected in the juvenile period of either C.hongkongensis or C.sikamea.This may be attributed to the very low survival of the tetrapioid larvae induced by this method,especially as most tetrapioid larvae died during the first three days.In summary,it is simple to directly induce tetrapioid C.hongkongensis and C.sikamea larvae by inhibiting the PB1 release of diploid zygotes,but the low survival rate makes it challenging to obtain viable juvenile tetrapioids.

Science Letters:Detection of Catabacter hongkongensis in polluted European water samples

The Catabacteriaceae is a new bacterial family aerobic, non-sporulating, Gram-positive coccobacillus that is with a unique member： Catabacter hongkongensis is a strictly anphylogenetically related to some clostridial clusters. Little is known of its epidemiology and environmental distribution, but the inclusion of its 16S rRNA gene sequence in GenBank has allowed it to be detected qualitatively. As a first approach for prospective surveys, a real-time polymerase chain reaction （PCR） procedure to identify C. hongkongensis has been developed. The presence of Catabacteriaceae in 29 water bodies subjected to possible human or animal impact has been investigated. Four of them were positive. The results confirm that highly polluted water can contain C hongkongensis.

盐度对近江牡蛎耗氧率、排氨率、O:N和吸收率的影响

以半现场流水槽法研究了不同盐度(5、10、15、20、25、30、35)对近江牡蛎(Crassostreahongkongensis)耗氧率、排氨率、O:N、吸收率的影响,并测定了盐度为20条件下牡蛎耗氧率和排氨率的周日变化。结果表明,近江牡蛎耗氧率、排氨率、O:N先随着盐度的升高而下降,在20左右降到最低,随着盐度继续上升,又升高;而近江牡蛎的吸收率先随着盐度的升高而升高,在20左右达到最高,然后随盐度升高而下降。根据数据得出耗氧率与盐度的拟合方程:y=0.0033x2-0.1161x+1.5523,R2=0.9018;排氨率与盐度:y=0.0001x2-0.0041x+0.0871,R2=0.9889;O:N与盐度:y=0.0016x3-0.0782x2+0.9051x+10.818,R2=0.955;吸收率与盐度:y=-0.0011x2+0.0399x+0.4393。R2=0.9453。一日内,8时、14时、19时、22时4个时间点,近江牡蛎耗氧率和排氨率变化较大,在14时最大,表明该时点其代谢活动最强。

人工诱导香港巨牡蛎雌核发育单倍体的研究

研究利用紫外线诱导香港巨牡蛎Crassostrea hongkongensis雌核发育的条件。结果表明,在强度为1820μW·(cm2·s)-1的紫外线(254nm)下分别照射精子0,5,10,15,20,25,30,35,40,45,50,55,60,70,80和90s后,照射30s的实验组能够保持较高的受精率(61.6%),该组的D形幼虫发生率为0,出现"Hertwig效应"。研究表明,在强度为1820μW·(cm2·s)-1的紫外线下照射精子30s是获得香港巨牡蛎雌核发育单倍体的适宜条件。研究还表明受精率和D形幼虫发生率随照射时间的增加而下降,遗传失活的精子与正常卵子受精后其胚胎发育至D形幼虫前期停止。经紫外线照射的精子授精后所产生的单倍体胚胎发育速度慢于正常受精所产生的二倍体胚胎,各照射组均出现非整倍体。

响应面法优化香港巨牡蛎蛋白酶解工艺的研究

以水解度为指标,比较了6种蛋白酶对香港巨牡蛎(Crassostrea hongkongensis)的水解效果。在单因素考察实验的基础上,采用Box-Behnken模型响应面设计,建立香港巨牡蛎蛋白酶解的二次回归模型,确定最佳的水解条件。实验结果表明,海产品专用蛋白酶对香港巨牡蛎蛋白的水解效果优于其他蛋白酶,该酶的最佳水解条件为:酶与底物比为5.93%,酶解时间为4.07 h,酶解温度为54.6℃。在此条件下,海产品专用蛋白酶酶解香港巨牡蛎蛋白的水解度达到25.73%±2.39%。本研究为香港巨牡蛎的开发和利用提供了理论依据。

近江牡蛎鳃细胞的原代培养

采用改良的DMEM(HG)培养基,建立了近江牡蛎(Crassostrea hongkongensis)鳃细胞体外培养技术,包括鳃组织块培养法和胰酶消化培养法。结果表明,组织块培养法接种6 h后,细胞开始从组织块中迁出,细胞形态较小,呈圆形、椭圆形或多边形,直径3～6μm。培养至3 d时,细胞在组织块周围形成生长晕。培养至6 d时可进行细胞传代,本次实验细胞已传至第6代。胰酶消化法接种约2 h后,细胞逐渐贴壁,从形态上主要分为两类,一类为小型细胞,形态为圆形、椭圆形或多边形,直径3～6μm,数量多,增殖速度较快;另一类为大型细胞,形态为圆形、椭圆形或多边形,直径10～20μm,部分细胞内部含有颗粒,数量较少,增殖速度较慢。培养至2 d时可进行细胞传代,传代培养物中的优势细胞皆为小型细胞。本次实验细胞已传至第6代。

香港巨牡蛎ISSR-PCR反应体系的建立及优化

以香港巨牡蛎(Crassostrea hongkongensis)基因组DNA为模板,采用正交设计及单因素比较试验,分别对Mg2+、dNTPs、引物、Taq DNA聚合酶和DNA模板浓度的PCR原料进行优化,并通过温度梯度PCR,筛选适宜的退火温度。确立了香港巨牡蛎的最适ISSR-PCR反应体系:25μL反应体系中含PCR 1×Buffer,2.5 mmol/L Mg2+、0.2 mmol/L dNTPs、0.2μmol/L引物、1.2 U Taq DNA聚合酶、20 ng DNA模板。反应程序为:94℃预变性5 min;94℃变性1 min,48～54℃(随引物而确定)退火1 min,72℃延伸1.5 min,35个循环;72℃延伸10 min。利用所建立的ISSR-PCR反应体系对香港巨牡蛎基因组DNA进行扩增,获得了清晰、重复性好、多态性高的DNA谱带,为进一步利用ISSR分子标记研究香港巨牡蛎的遗传多样性奠定了基础。

牡蛎体内及其养殖水体中细菌耐药性研究

为进一步了解牡蛎体内外细菌的耐药性,对分离自阳江的香港牡蛎(Crassostrea hongkongensis)养殖水体及其内脏团的637个异养细菌进行了10种抗生素的药敏试验。采用纸片扩散法(Kirby-Bauer,简称K-B),参照NCCLS抗生素敏感试验操作标准,研究细菌的耐药概况。结果显示:健康牡蛎体内细菌对恩诺沙星和庆大霉素耐药率较低,发病牡蛎体内细菌对庆大霉素耐药率最低,次之为复合磺胺和恩诺沙星;水体细菌对氯霉素、恩诺沙星、环丙沙星耐药率较低,水体及体内细菌对呋喃唑酮耐药率最高,而对卡那霉素及青霉素耐药率则波动较大。结果还显示,不同来源的受试菌株多重耐药状况严重,来源于健康和病牡蛎的菌株在7月份的多重耐药率达到峰值,分别为66.7%和64.3%,养殖水体细菌多重耐药率峰值出现在4月份,最高达76.7%。总体来看,源于牡蛎养殖环境水体及其牡蛎体内的异养菌多重耐药比例较高,值得引起注意。而受试菌株对不同药物的抗性存在较大差异,且异养细菌对呋喃唑酮的耐药率明显高于对其它受测抗生素的耐药率。从时间动态上看,水体菌耐药率峰值出现于4月份,而牡蛎体内菌群耐药率峰值出现于7月份。

盐度胁迫对香港牡蛎部分生化指标的影响

为探究盐度胁迫下香港牡蛎(Crassostrea hongkongensis)生化指标的变化规律,了解盐度适应过程中牡蛎的代谢机制,本研究以盐度0、8、16、32、40 为胁迫盐度,以正常海水(盐度24)为对照,开展香港牡蛎对盐度胁迫的响应研究。结果显示,各实验盐度组糖原含量在盐度胁迫0~8 h 内下降,且盐度胁迫幅度越大糖原含量降幅越大,胁迫8 h 后则无明显的变化规律。腺苷酸激活的蛋白激酶(AMPK)、组蛋白/蛋白去乙酰化酶(SIRT1)和Na^+/K^+-ATP 酶活力的变化规律相似:在0~8 h 内,各实验组酶活力均急剧下降(SIRT1 上升);8~48 h 内,酶活力上升;48~120 h 内,酶活力逐渐趋于平稳状态,总体表现为盐度越高,酶活力越强,并与胁迫前有明显差异,且盐度胁迫幅度越高,差异越明显。总抗氧化能力(T-AOC)总体表现出高盐胁迫下随时间的增加而升高,低盐胁迫下随时间的增加而降低,且盐度胁迫幅度越大, T-AOC 活力的变化幅度越大。实验结果初步表明,香港牡蛎糖原含量与渗透压调节存在一定的关系, AMPK、SIRT1、Na^+/K^+-ATP 酶活力及T-AOC 均与渗透压调节密切相关,且在高盐胁迫下随时间的增加而升高,低盐胁迫下随时间的增加而降低。