苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

Construction of brewing-wine Aspergillus oryzaepyrG- mutant bypyrG gene deletion and its application in homology transformation

pyrG- host cells are indispensable for pyrG- based transformation system. Isolations ofpyrG- host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG- mutant by sitedirected mutation of pyrG gene deletion which would be used as a host for further transformation, pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to con- struct pyrG- mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating thatpyrG was completely excised. The ApyrG mutants were applied as pyrG- host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production ofA. Oryzae. The xdh disrup- tion mutants were efficiently constructed by transforming a pMD-pyrC-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Axdh mutant was three times as much as that of the ApyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG- host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding ofA. Oryzae.     

Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins(RED) production

Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g~(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.     

Characterization of the Rice Floral Organ Number Mutant fon3

A spontaneous rice mutant named floral organ number 3 (fon3) had major mutations in floral organ numbers. Genetic analysis indicated that fort3 acted as a single recessive gene. Microscopic observation showed that the number of floral organs infon3 increased centripetally. For example, the number of pistils was the more frequently increased than organs in the outer whorls. Homeotic conversion of lodicules and glumes into palea/lemma-like organs was observed in some flowers. Scanning electron microscopy observation showed that the size of flower meristems was maintained the same or similar until the lemma primordium started to differentiate, at which time the floral meristem became enlarged, suggesting abnormal development of the inner whorls of rice florets. The relationship of fort3 with other similar rice mutants is discussed.     

Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.     

Partial rescue of pos5 mutants by YEF1 and UTR1 genes in Saccharomyces cerevisiae

Three NAD kinase homologs,encoded by UTR1,POS5 and YEF1 genes,are found in the yeastSaccharomyces cerevisiae and proven to be important sources of NADPH for the cell.Pos5p,existing in themitochondrial matrix,is critical for higher temperature endurance and mitochondrial functions,such asglycerol usability and arginine biosynthesis.Through constructing the high-copy expression plasmids ofYEF1 and UTR1,which contained the green fluorescent protein reporter tag at their 3' terminus,and introducingthem into POS5 gene deletion mutants(i.e.pos5,utr1pos5,yef1pos5 and utr1yef1pos5),the high-copy YEF1and UTR1 plasmids carrying transformants for pos5 mutants were obtained.Their temperature sensitivityand growth phenotype on media with glycerol as the sole carbon source,or on media without arginine,werechecked.Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity ofpos5 mutants by the high-copy YEF1 gene,and of glycerol growth defect and temperature sensitivity by thehigh-copy UTR1 gene,which confirmed the potential supplying ability of Yeflp and Utrlp for mitochondrialNADP(H)and implied the weak transport of NADP from cytosol to mitochondria.However,even throughthe green fluorescent protein reporter label,the subcellular localization of Yef1p and Utr1p in yeast cells couldnot be observed,which indicated the low expression level of these two NAD kinase homologs.     

SaliCylic Acid-Altering Arabidopsis Mutants Response to Cd Stress

To evaluate the role of endogenous SA in plant response to Cd stress,Arabidopsis wild type(Columbia)and its SA-altering mutants snc1 (suppressor of npr1-1, constitutive) with high SA level, nahG(tansgenic line)with low SA level and npr1-1(non-expressor of PR gene)with SA signaling blockage were used in this study. Results showed that a greater growth inhibition occurred in snc1,while a less inhibition was observed in nahG and npr1-1 plants. Although the anti-oxidative enzymes SOD and POD increased upon Cd exposure,they were insufficient to remove oxidative stress,especially in snc1 plants. The accumulations of soluble sugar and proline in the tested plants were positively related to their tolerance to Cd stress.