Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.     

用少量样本进行抑制性消减杂交

利用根据cap-finder方法建立的全长cDNA合成技术,扩增获得了恒河猴着床点子宫内膜组织表达mRNA的双链cDNA,通过抑制性消减杂交,成功地构建了恒河猴着床点消减文库.随机挑选文库中的阳性克隆,经点杂交证明27％为着床点差异表达的克隆.由此表明抑制性消减杂交结合cap-finder扩增全长cDNA的方法,可以有效地从少量而珍贵的样本中获得高质量的消减文库.     

Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases

Soybean rust caused by Phakopsora pachyrhizi Sydow is a devastating foliar disease that has spread to most soybean growing regions throughout the world, including the USA. Four independent rust resistance genes, Rpp1–Rpp4, have been identified in soybean that recognize specific isolates of P. pachyrhizi. A suppressive subtraction hybridization (SSH) complementary DNA (cDNA) library was constructed from the soybean accession PI200492, which contains Rpp1, after inoculation with two different isolates of P. pachyrhizi that result in susceptible or immune reactions. Both forward and reverse SSH were performed using cDNA from messenger RNA pooled from 1, 6, 12, 24, and 48 h post-inoculation. A total of 1,728 SSH clones were sequenced and compared to sequences in GenBank for similarity. Microarray analyses were conducted on a custom 7883 soybean-cDNA clone array encompassing all of the soybean-rust SSH clones and expressed sequence tags from four other soybean cDNA libraries. Results of the microarray revealed 558 cDNA clones differentially expressed in the immune reaction. The majority of the upregulated cDNA clones fell into the functional category of defense. In particular, cDNA clones with similarity to peroxidases and lipoxygenases were prevalent. Downregulated cDNA clones included those with similarity to cell-wall-associated protein, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction between an isolate of dark-septate fungi and its host plant <Emphasis Type="Italic">Saussurea involucrata</Emphasis>

A dark-septate endophytic (DSE) fungus EF-M was isolated from the roots of an alpine plant Saussurea involucrata Kar. et Kir. ex Maxim. The fungus was identified by sequencing the PCR-amplified rDNA 5.8S gene and ITS regions. The sequence was compared with similar sequences in the GenBank, and results showed that EF-M was congeneric to Leptodontidium. Resynthesis study was conducted to clarify the relationship between the root endophyte EF-M and the host plant S. involucrata using the material grown in sterile culture bottle. In roots recovered 6 weeks after inoculation, epidermal cells were colonized by intercellular and intracellular hyphae and “microsclerotia” formed within individual cells in the epidermis layers. However, hyphae did not invade the cortex and the stele. There were no profound effects of endophyte EF-M on plant root development, but significant differences were detected in plant height and shoot dry weight between the treatments. The present study is the first report hitherto on DSE fungi in S. involucrata.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

Expressed sequence tags from persimmon at different developmental stages

Persimmon (Diospyros kaki Thunb.) is an important fruit in Asian countries, where it is eaten as a fresh fruit and is also used for many other purposes. To understand the molecular mechanism of fruit development and ripening in persimmon, we generated a total of 9,952 expressed sequence tags (ESTs) from randomly selected clones of two different cDNA libraries. One cDNA library was derived from fruit of “Saijo” persimmon at an early stage of development, and the other from ripening fruit. These ESTs were clustered into 6,700 non-redundant sequences. Of the 6,700 non-redundant sequences evaluated, the deduced amino acid sequences of 4,356 (65%) showed significant homology to known proteins, and 2,344 (35%) showed no significant similarity to any known proteins in Arabidopsis databases. We report comparison of genes identified in the two cDNA libraries and describe some putative genes involved in proanthocyanidin and carotenoid synthesis. This study provides the first global overview of a set of genes that are expressed during fruit development and ripening in persimmon.     

Cloning,sequence analysis,and prokaryotic expression of cDNA encoding a putative non-specific lipid-transfer protein from the bracts of dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis> Baill)

The 967-bp cloneP1A5 was isolated from a suppression subtractive hybridization cDNA library of dovetree bracts (Davidia involucrata Baill.). A complete cDNA of 1047 bp was obtained via 5-RACE (5′Rapid Amplification of cDNA End) techniques, using the gene-specific primer P1A5-1. Northern blot analyses showed that the gene was predominantly expressed in the bracts, while the blotting signal from the leaves was weak. Its deduced amino acid sequence was most highly homologous to the lipid-transfer protein 3 precursor isolated from upland cotton, the lipid-transfer protein SDi-9 from the common sunflower, and the non-specific lipid-transfer protein precursor allergen from sweet cherry. It also had features in common with plant nsLTPs (non-specific lipid-transfer proteins), including eight conserved cysteine residues, a high isoelectric point (8.9), and a lack of tryptophans. The deduced amino acid sequence had two transmembrane helices -- the first from Position 5 (Gly) to Position 35 (Val); the second, from Position 28 (Ala) to Position 46 (Leu). A cleavage site for the putative signal peptide was predicted to occur between Positions 28 (Ala) and 29 (Ala). Therefore, the putative mature form of the protein would comprise 92 amino acids, with a molecular weight of 9.2 kD. All these results provide compelling evidence that theP1A5 clone belongs to the nsLTP1 gene family, thus being named theP1A5 putative nsLTPI gene. This is the first nsLTP gene reported from Davidiaceae. The nucleotide sequence data reported here has been released in the GenBank, EMBL, and DDBJ Nucleotide Sequence Databases, under the accession number AY059472.     

Screening and comparison of differentially expressed genes between one MDR-TB strain and the virulent M. tuberculosis H37Rv

To screen and compare the differentially expressed genes between one MDR-TB strain separated from one child patient and the virulent Mycobacterium tuberculosis H37Rv, suppression subtractive hybridization (SSH) technology was used to build a library of cDNAs that were differentially expressed in the MDR and H37Rv. From this cDNA library, genes that were expressed in the MDR-TB but not in the H37Rv were selected for gene sequencing and homology analysis; 113 positive clones were obtained, their cDNA fragments were sequenced, and homology analysis was performed. Four novel sequences were identified. The results provide a partial list of genes differentially expressed in MDR-TB and four novel genes were found. Identification of these genes may contribute to our understanding of MDR-TB development.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases

A cDNA library from freshly isolated protoplasts was differentially screened using cDNAs from mesophyll cells, stressed leaf strips and cell suspension cultures. One of the selected clones, 6P229, turned out to encode a putative polypeptide showing homology to the btuE periplasmic protein of Escherichia coli and to animal selenium-dependent glutathione peroxidases. A major difference was that the putative selenocysteine in the active site was not encoded by the termination codon TGA. The 6P229 gene was found to be expressed in germinating seeds, in apex and in flowers, as well as in stressed tissues. This pattern of expression would be consistent with a key role in cellular metabolism such as defense against oxidative stresses.     

不同色彩珙桐叶片和苞片解剖结构及色素含量比较研究

以生长于同一生境下的粉红珙桐(粉红色叶片、苞片)与普通珙桐(绿色叶片、白色苞片)为试材,对比两种色彩珙桐叶片/苞片解剖结构和色素含量的差异,以揭示珙桐色彩转变的规律。结果显示：(1)两种珙桐叶片均属于异面叶类型,栅栏组织由一层长柱形细胞整齐排列而成,海绵组织排列疏松,部分粉红叶片的上表皮细胞向外凸起,绿叶无此现象;粉红叶片的总厚度及其表皮角质层、栅栏组织和海绵组织厚度都高于绿叶,而表皮较薄。(2)两种珙桐苞片均无栅栏组织和海绵组织的分化,粉红苞片上表皮细胞明显隆起,上表皮角质层增厚,而下表皮变薄。(3)粉红叶片的类黄酮、花色苷含量分别是绿色叶片的1.52倍、3.67倍,两者的光合色素含量无显著差异,但粉红叶片的叶绿素a/b值比绿色叶低很多;粉红苞片花色苷含量显著高于白色苞片,而两者类黄酮含量差异不大。研究表明,花色苷是珙桐叶片和苞片色彩转红的直接因素,类黄酮有助于叶片呈红色;粉红珙桐叶片/苞片的解剖结构发生了一定变化,对光能的利用效率更高,对阴湿环境的适应性增强。