Cloning And Sequencing
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2021 ◽  
Vol 12 ◽  
Yan Men ◽  
Ji-Rui Li ◽  
Hai-Lin Shen ◽  
Yi-Ming Yang ◽  
Shu-Tian Fan ◽  

In the past decade, progress has been made in sex determination mechanism in Vitis. However, genes responsible for sexual differentiation and its mechanism in V. amurensis remain unknown. Here, we identify a sex determination candidate gene coding adenine phosphoribosyl transferase 3 (VaAPRT3) in V. amurensis. Cloning and sequencing of the VaAPRT3 gene allowed us to develop a molecular marker able to discriminate female individuals from males or hermaphrodites based on a 22-bp InDel. Gene expression and endogenous cytokinin content analysis revealed that the VaAPRT3 gene is involved in sex determination or, to be precise, in female organ differentiation, through regulating cytokinin metabolism in V. amurensis. This study enlarged the understanding of sex determination mechanism in the genus Vitis, and the sex marker could be used as a helpful tool for sexual identification in breeding programs as well as in investigation and collection of V. amurensis germplasms.

2021 ◽  
Vol 1 (1) ◽  
Isaiah Catalino M. Pabuayon ◽  
Jiafu Jiang ◽  
Hongjia Qian ◽  
Jung-Sung Chung ◽  
Huazhong Shi

AbstractSoil salinity severely hampers agricultural productivity. Under salt stress, excess Na+ accumulation causes cellular damage and plant growth retardation, and membrane Na+ transporters play central roles in Na+ uptake and exclusion to mitigate these adverse effects. In this study, we performed sos1 suppressor mutant (named sup) screening to uncover potential genetic interactors of SOS1 and additional salt tolerance mechanisms. Map-based cloning and sequencing identified a group of mutants harboring dominant gain-of-function mutations in the vacuolar Na+/H+ antiporter gene AtNHX1. The gain-of-function variants of AtNHX1 showed enhanced transporter activities in yeast cells and increased salt tolerance in Arabidopsis wild type plants. Ion content measurements indicated that at the cellular level, these gain-of-function mutations resulted in increased cellular Na+ accumulation likely due to enhanced vacuolar Na+ sequestration. However, the gain-of-function suppressor mutants showed reduced shoot Na+ but increased root Na+ accumulation under salt stress, indicating a role of AtNHX1 in limiting Na+ translocation from root to shoot. We also identified another group of sos1 suppressors with loss-of-function mutations in the Na+ transporter gene AtHKT1. Loss-of-function mutations in AtHKT1 and gain-of-function mutations in AtNHX1 additively suppressed sos1 salt sensitivity, which indicates that the three transporters, SOS1, AtNHX1 and AtHKT1 function independently but coordinately in controlling Na+ homeostasis and salt tolerance in Arabidopsis. Our findings provide valuable information about the target amino acids in NHX1 for gene editing to improve salt tolerance in crops.

2021 ◽  
Vol 11 (1) ◽  
Dmitrii Sliadovskii ◽  
Tatyana Ponomareva ◽  
Maxim Molchanov ◽  
Irina Pozdnyakova-Filatova ◽  
Maria Timchenko ◽  

AbstractCrustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40–50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the $$\beta $$ β -elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

2021 ◽  
Vol 11 (1) ◽  
Josephine Dubois ◽  
Georg Sczakiel

AbstractNumerous indirect and in silico produced evidences suggest circular RNAs (circRNA) in mammals while thorough experimental proofs of their existence have rarely been reported. Biological studies of circRNA, however, should be based on experimentally verified circRNAs. Here, we describe the identification of two circRNAs originating from the gene locus of the translocation associated membrane protein 1 (TRAM1). Linear and potentially circular TRAM1-specific transcripts were identified in a transcriptome analysis of urine RNA of bladder cancer (BCa) patients versus healthy donors. Thus, we first focused on the topology of TRAM1-specific transcripts. We describe conclusive experimental evidence for the existence of TRAM1-specific circRNAs in the human BCa cell lines ECV-304 and RT-4. PCR-based methodology followed by cloning and sequencing strongly indicated the circular topology of two TRAM1 RNAs. Further, studies with exon fusion sequence-specific antisense oligonucleotides (asON) and RNase H as well as studies in the use of RNase R contribute to conclusive set of experiments supporting the circular topology of TRAM1 transcripts. On the biological side, TRAM1-specific circRNAs showed low expression levels and minor differences in BCa cell lines while linear TRAM1 transcripts displayed down-regulated expression in the higher cancer stage model ECV-304 versus more differentiated RT-4 cells.

2021 ◽  
Vol 26 (3) ◽  
pp. 142
Anissa Utami ◽  
Pamela Apriliana ◽  
Yudi Kusnadi ◽  
Dewi S. Zilda ◽  
Zidny Ilmiah ◽  

We investigated the biosynthetic potential of soil-associated actinobacteria originating from Indonesia, identified as Streptomyces luridus and as Streptomyces luteosporeus. Antimicrobial assays indicated inhibitory activity by both strains against the pathogen Pseudomonas aeruginosa, with S. luteosporeus particularly inhibiting the growth of Bacillus subtilis. PCR-amplification, cloning, and sequencing of ketosynthase (KS) domains of type I modular polyketide (PKS-I) and adenylation (AD) domains of non-ribosomal peptide synthetase (NRPS) indicated the diversity of KS and AD domains derived from both Indonesian Streptomyces. Further phylogenetic analysis showed that KS domains from the subclass cis-AT PKS can be classified as being a part of a loading module or an extension module, along with their predicted substrate specificity. The results suggest that both strains are a potential source of novel biosynthetic pathways. This genetic analysis approach can be used as a fast guide to obtain insight into natural product biosynthetic gene diversity in microorganisms.

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 457
Emma L. Betts ◽  
Sumaiya Hoque ◽  
Lucy Torbe ◽  
Jessica R. Bailey ◽  
Hazel Ryan ◽  

(1) Background: Blastocystis is a microbial eukaryote inhabiting the gastrointestinal tract of a broad range of animals including humans. Several studies have shown that the organism is associated with specific microbial profiles and bacterial taxa that have been deemed beneficial to intestinal and overall health. Nonetheless, these studies are focused almost exclusively on humans, while there is no similar information on other animals. (2) Methods: Using a combination of conventional PCR, cloning and sequencing, we investigated presence of Blastocystis along with Giardia and Cryptosporidium in 16 captive water voles sampled twice from a wildlife park. We also characterised their bacterial gut communities. (3) Results: Overall, alpha and beta diversities between water voles with and without Blastocystis did not differ significantly. Differences were noted only on individual taxa with Treponema and Kineothrix being significantly reduced in Blastocystis positive water voles. Grouping according to antiprotozoal treatment and presence of other protists did not reveal any differences in the bacterial community composition either. (4) Conclusion: Unlike human investigations, Blastocystis does not seem to be associated with specific gut microbial profiles in water voles.

O. O. Ishchenko ◽  
V. V. Kozub ◽  
I. I. Panchuk

Aim. 5S ribosomal DNA (5S rDNA) represents a universal model for studying the evolution of repeated sequences in eukaryotic organisms. Taking into account that this region of the genome still remains almost undescribed in species of the family Sapindaceae, we investigated the molecular organization of a repeated unit of 5S rDNA in a member of this family, Litchi chinensis. Methods. PCR amplification, cloning and sequencing of 5S rDNA. Results. It was found that the length of the repeated unit of the 5S rDNA of L. chinensis is 321 -323 bp. The level of intragenomic similarity of 5S rDNA repeats is 87.1 %. Potential external elements of the RNA polymerase III promoter, which are localized in IGS, differ from those described for members of other families of angiosperms. Conclusions. In the genome of L. chinensis, at least two classes of 5S rDNA repeats are present, which differ in the sequence of external promoter elements. Keywords: 5S rDNA, molecular evolution, Litchi chinensis, Sapindaceae.

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