Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis： Effects of Cultivation Parameters

The green alga, Haematococcus pluvlalis Flotow is used as a source of the ketocarotenoid astaxanthin for application in fish aquaculture, pharmaceutical and cosmetic industries. Ceils of the green alga were induced by the application of different light and starvation conditions to evaluate the effect in astaxanthin accumulate. The conditions used for the Induction were high light intensity （170 μmol·m^-2·s^-1）, iron starvation, sulfur starvation and phosphate starvation. The results show that stresses applied in culture, which interfere with cell division, trigger the accumulation of astaxanthin. Notably, sulfur starvation results in a massive accumulation of this commercially important carotenoid.     

低温胁迫下不同光照条件对锦熟黄杨抗氧化酶活性的影响

研究了锦熟黄杨（Buxus sempervirens L.）在低温胁迫下不同光照条件（12 h光照/12 h黑暗、24 h全光、24 h全黑）对其抗氧化酶活性的影响,结果表明：低温不同光照条件下细胞膜透性和丙二醛含量均高于对照（20℃ 12 h光照/12 h黑暗）,细胞膜透性和丙二醛含量在5℃ 24 h光照条件下最大;低温胁迫下SOD活性高于对照,并在5℃ 24 h光照条件下达到最高值;CAT的活性仍维持较高水平,5℃ 12 h光照/12 h黑暗显著高于对照及其它处理;在低温有光照条件下,POD活性升高,黑暗条件下POD活性低于对照。低温胁迫下POD、SOD和CAT的活性均呈上升趋势,可能是植株具有较强抗性的原因。     

显脉金花茶的光合生理特性研究

采用LI-6400便携式光合测定系统(Li-Cor Inc., USA)对显脉金花茶光合特性进行研究。结果表明：(1)在夏季,显脉金花茶叶片的P_n日变化呈单峰曲线,最高峰出现在中午11：00。其最大净光合速率（P_max）为3.81 μmol·m^-2·s^-1、光饱和点（LSP）为459.9 μmol·m^-2·s^-1、光补偿点（LCP）为6.9 μmol·m^-2·s^-1。显脉金花茶的光饱和点和光补偿点都比较低,表明其是一种阴生植物。（2）在控制光照强度和温度的条件下,CO₂浓度小于800 μmol·mol^-1,P_n几乎呈直线上升,升高CO₂浓度可使显脉金花茶的净高合速率增大,提高了叶片对光能的利用率。其叶片CO₂饱和点(CSP)大约在1 200 μmol·mol^-1左右,CO₂的补偿点（Г^*）为70.1 μmol·mol^-1,最大羧化速率（V_cmax）为17.5 μmol·m^-2·s^-1,最大电子传递速率（J_max）为40 μmol·m^-2·s^-1。     

Detection of a new mutation (T1140C) in a patient with Hunter syndrome from Guangdong,China

This study identified mutations of the idumate-2-suffatase (IDS) gene in a patient with Hunter syndrome,and established a basis for the diagnosis of the prenatal gene of Hunter syndrome.Urine glyeosaminoglycan (GAG) assay was used to make the preliminary diagnosis of mucopolysaccharidosis type H.Polymerase chain reaction (PCR) from dried blood spots and DNA sequencing were applied to analyze hotspot mutations in exons 9,3 and 8 of the IDS gene in the proband and his parents.A new missense mutation (T1140C) in exon 8 of the IDS gene was found by using DNA sequencing.This mutation caused a substitution of codon 339 from CTA (leucine) to CCA (praline).The patient is a hemizygote,and his mother is a heterozygote.The new missense mutation results in a change in the primary and tertiary structure of the IDS protein.It is possible that this mutation severely impairs enzymatic activity and is the underlying basis for the pathology seen in this patient with Hunter syndrome.     

The effect of ultraviolet-depleted light on the flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea

An early investigation at the Biosphere-2 Laboratory, an artificial ecosystem in the Arizona desert, had shown that the flavonoid content of cacti grown in glass-filtered solar light was lower than of cacti grown in normal solar light. This was attributed to the absence of ultraviolet (UV) radiation, which is required for flavonoid biosynthesis. In this study, two species of Opuntia cacti were grown in solar and UV-depleted light, and their flavonol contents of different tissues were determined by HPLC. O. wilcoxii, previously raised in the absence of UV light, was exposed to normal solar light. The flavonol content of young O. wilcoxii pads was 28-fold higher when grown in solar light as compared to UV-depleted light. The flavonol contents of mature outer tissues were only slightly higher. O. violacea, previously raised in solar light, was also maintained in the same UV-depleted artificial ecosystem. The flavonol content after hydrolysis of outer tissues was similar, whether grown in solar light or UV-depleted light. We attribute these responses to different biosynthetic and metabolic rates of young vs. mature plant tissues; slow-growing mature tissues neither produce nor metabolize compounds as quickly as immature tissues. These findings indicate that artificial ecosystems can influence the production of natural products in cultivated plants.     

不同氮素供应下水稻酚类物质代谢关键酶基因差异表达

运用实时荧光定量PCR技术探讨了不同供氮条件下强化感与弱化感水稻苯丙烷代谢途径中9个关键酶基因的表达差异。结果表明,与正常氮素供应相比,低氮胁迫引起强化感水稻‘P1312777’中与酚类代谢途径相关的9个关键酶基因表达量均上调,表达量增幅在1.9～5.4倍之间,且以PAL基因上调倍数最大。而弱化感水稻‘Lemont’则相反,只有2个基因(苯丙氨酸裂解酶基因和肉桂酰CoA基因)表达上调,但上调倍数分别是强化感水稻对应的基因的22%和74%,其余的7个基因表达均下调,降幅在29%～72%之间,表明低氮胁迫诱发的水稻化感抑草能力增强与其体内酚类物质合成代谢增强有关。     

用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants）,这些突变体中包含大量的限制性内切酶位点,选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段,然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C,T两个碱基,校正了阅读框架,获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性,这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。     

采后光照条件对白凤桃果实品质和挥发性香气物质形成的影响

研究了白凤桃果实贮藏过程中光照条件对果实成熟的影响。在7月12日(未熟期)和7月16日(硬熟期)采收果实,分别贮藏在光条件(白色荧光灯照明,果顶部光强为80μmol m~(-2)s~(-1))和暗条件中,室温均为25℃。硬熟期采收果实贮藏在光条件下,达到完熟期时,乙烯生成量较低。果肉的硬度在各个采收期,各种贮藏条件下均没有差别。光条件贮藏果实中花青苷含量较高。未熟期采收果实贮藏在光条件下时,可溶性固形物含量增加较多。光条件贮藏果实中天冬酰胺的下降比暗贮藏果实中更多。各时期采收的果实中,在光下贮藏时,果肉和果皮γ-癸内酯和γ-十二内酯的含量都明显增加。以上结果表明,白凤桃果实采收后在光下贮藏,可以明显改善果实的品质。