Jiao Liu

Genome-Wide Identification, 3D Modeling, Expression and Enzymatic Activity Analysis of Cell Wall Invertase Gene Family from Cassava (Manihot esculenta Crantz)

The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

Genome-Wide Identification, Expression, and Activity Analysis of Alkaline/Neutral Invertase Gene Family from Cassava (Manihot esculenta Crantz)

Alkaline/neutral invertase comprises a family of enzymes that catalyze the irreversible hydrolysis of sucrose into glucose and fructose. They play a crucial role in carbohydrate partitioning and developmental processes of plants. In this study, we genome-wide identified 11 alkaline/neutral invertase family genes (MeNINVs) from the cassava genome. Based on phylogeny, MeNINVs could be classified into α and β groups. The α group genes are encoded by six exons (MeNINV1, 6, 7, 8, and 10) or four exons (MeNINV9), whereas the β group genes are encoded by four exons (MeNINV2, 3, 4, and nINV1) or five exons (MeNINV5). Their catalytic residues of motifs 2 and 4 for sucrose hydrolysis locate in all MeNINVs. qRT-PCR analysis has demonstrated that all MeNINVs are highly expressed in floral organs. During tuber development, MeNINV1, 6, 10, and nINV1 are strongly expressed in source tissues (leaves), and MeNINV1 and nINV1 also play an important role in sink organs (tuber). Activity analysis of alkaline/neutral invertase and determination of sucrose content in source and sink organs of cassava indicates that lower efficiency of sucrose hydrolysis in source organs (leaves) and higher efficiency in sink organs (tuber) are beneficial sucrose to transport from source to sink organs.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava

Hexokinase (HXK) proteins play important roles in catalyzing hexose phosphorylation and sugar sensing and signaling. To investigate the roles of HXKs in cassava tuber root development, seven HXK genes (MeHXK1–7) were isolated and analyzed. A phylogenetic analysis revealed that the MeHXK family can be divided into five subfamilies of plant HXKs. MeHXKs were clearly divided into type A (MeHXK1) and type B (MeHXK2–7) based on their N-terminal sequences. MeHXK1–5 all had typical conserved regions and similar protein structures to the HXKs of other plants; while MeHXK6–7 lacked some of the conserved regions. An expression analysis of the MeHXK genes in cassava organs or tissues demonstrated that MeHXK2 is the dominant HXK in all the examined tissues (leaves, stems, fruits, tuber phloems, and tuber xylems). Notably, the expression of MeHXK2 and the enzymatic activity of HXK were higher at the initial and expanding tuber stages, and lower at the mature tuber stage. Furthermore, the HXK activity of MeHXK2 was identified by functional complementation of the HXK-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). The gene expression and enzymatic activity of MeHXK2 suggest that it might be the main enzyme for hexose phosphorylation during cassava tuber root development, which is involved in sucrose metabolism to regulate the accumulation of starch.

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1–7) were identified, and MeFRK1–6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK 1, 2, 6, 7) and β (MeFRK 3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1–6 were similar, folding into a catalytic domain and a β-sheet ‘‘lid” region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1–6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.

Cloning, 3D Modeling and Expression Analysis of Three Vacuolar Invertase Genes from Cassava (Manihot Esculenta Crantz)

Vacuolar invertase is one of the key enzymes in sucrose metabolism that irreversibly catalyzes the hydrolysis of sucrose to glucose and fructose in plants. In this research, three vacuolar invertase genes, named MeVINV1-3, and with 653, 660 and 639 amino acids, respectively, were cloned from cassava. The motifs of NDPNG (β-fructosidase motif), RDP and WECVD, which are conserved and essential for catalytic activity in the vacuolar invertase family, were found in MeVINV1 and MeVINV2. Meanwhile, in MeVINV3, instead of NDPNG we found the motif NGPDG, in which the three amino acids GPD are different from those in other vacuolar invertases (DPN) that might result in MeVINV3 being an inactivated protein. The N-terminal leader sequence of MeVINVs contains a signal anchor, which is associated with the sorting of vacuolar invertase to vacuole. The overall predicted 3D structure of the MeVINVs consists of a five bladed β-propeller module at N-terminus domain, and forms a β-sandwich module at the C-terminus domain. The active site of the protein is situated in the β-propeller module. MeVINVs are classified in two subfamilies, α and β groups, in which α group members of MeVINV1 and 2 are highly expressed in reproductive organs and tuber roots (considered as sink organs), while β group members of MeVINV3 are highly expressed in leaves (source organs). All MeVINVs are highly expressed in leaves, while only MeVINV1 and 2 are highly expressed in tubers at cassava tuber maturity stage. Thus, MeVINV1 and 2 play an important role in sucrose unloading and starch accumulation, as well in buffering the pools of sucrose, hexoses and sugar phosphates in leaves, specifically at later stages of plant development.

Isolation and Characterization of Ftsz Genes in Cassava

The filamenting temperature-sensitive Z proteins (FtsZs) play an important role in plastid division. In this study, three FtsZ genes were isolated from the cassava genome, and named MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2, respectively. Based on phylogeny, the MeFtsZs were classified into two groups (FtsZ1 and FtsZ2). MeFtsZ1 with a putative signal peptide at N-terminal, has six exons, and is classed to FtsZ1 clade. MeFtsZ2-1 and MeFtsZ2-2 without a putative signal peptide, have seven exons, and are classed to FtsZ2 clade. Subcellular localization found that all the three MeFtsZs could locate in chloroplasts and form a ring in chloroplastids. Structure analysis found that all MeFtsZ proteins contain a conserved guanosine triphosphatase (GTPase) domain in favor of generate contractile force for cassava plastid division. The expression profiles of MeFtsZ genes by quantitative reverse transcription-PCR (qRT-PCR) analysis in photosynthetic and non-photosynthetic tissues found that all of the MeFtsZ genes had higher expression levels in photosynthetic tissues, especially in younger leaves, and lower expression levels in the non-photosynthetic tissues. During cassava storage root development, the expressions of MeFtsZ2-1 and MeFtsZ2-2 were comparatively higher than MeFtsZ1. The transformed Arabidopsis of MeFtsZ2-1 and MeFtsZ2-2 contained abnormally shape, fewer number, and larger volume chloroplasts. Phytohormones were involved in regulating the expressions of MeFtsZ genes. Therefore, we deduced that all of the MeFtsZs play an important role in chloroplast division, and that MeFtsZ2 (2-1, 2-2) might be involved in amyloplast division and regulated by phytohormones during cassava storage root development.

Cloning and Analysis of Vacuolar Invertase Gene (MeVINV2) Promoter from Cassava (Manihot Esculenta Crantz)

Vacuolar invertases play a vital role in the progress of cassava tuber roots starch accumulation. In order to study the regulating mechanism of cassava vacuolar invertases, the promoter of cassava vacuolar invertase 2 (MeVINV2) was isolated using the PCR amplification approach, starting with a part of coding sequences. Sequencing result showed that 47 bp MeVINV2 gene CDS sequence and 1242 bp potential promoter sequence was obtained. PlantCARE analysis revealed that the MeVINV2 gene promoter contains typical eukaryotic elements CAAT box and TATA box, and also several light-responsive elements and stress-responsive elements. These cis-acting regulatory elements might be associated to the vacuolar invertase gene function of cassava starch accumulation and biological stress defense.

Cloning of MeCWINV3 Promoter from Cassava and Transient Expression Analysis in Tobacco

In order to study the inducement pattern and regulating mechanism of MeCWINV3 in Cassava. An 1160 bp promoter region upstream of the MeCWINV3 gene (GenBank Accession No. KC905170) was isolated from Cassava (Manihot esculenta) genomic DNA using PCR methods. Sequence analysis found that it contains typical TATA box and CAAT box, and several cis-acting elements that related plant stress responses, such as ABRE, ARFAT, GAREAT, MYB and MYC transcription factors. Furthermore, transient expression in transgenic tobacco was analyzed by inserting upstream of GUS gene in expressing vector. The results showed that GUS was mainly expressed in tobacco veins. This will be the basis for further investigating the function of the MeCWINV3 gene promoter.

Expression of CP:CBF3-35S:ICE1 enhances low temperature tolerance in transgenic Arabidopsis

We have constructed a vector pCAMBIA1300-CP:CBF3-35S:ICE1 and transformed into Arabidopsis. Results of PCR proved that the target genes had integrated into Arabidopsis genome. Transgenic Arabidopsis showed a bit slow growth, earlier flowering, but normal at other phenotype under 22°C with 8 h daily lights. In vitro low temperature stress tests showed that the transgenic lines were survival while the wild type was nearly dead. The transgenic plants also showed an increased proline content, SOD and POD activities under low temperature stress. The phenotype and physical evidence indicated that expression of CP:CBF3-35S:ICE1 under low temperature enhances the cold tolerance in transgenic plants.

Optimization of the Heterologous Expression of Sesuvium portulacastrum Betaine Aldehyde Dehydrogenase in Escherichia coli

A full-length sequence coding for a betaine aldehyde dehydrogenase gene from S. portulacastrum was cloned into expression vector pGEX-4T-1, and named pGEX-4T-SpBADH. The GST-SpBADH fusion protein was expressed and the expression conditions were optimized. Through the research on optimization of expression the concentration of IPTG, concentration of bacterium, induction time and temperature and so on, the results showed, the expression of GST-SpBADH increased accompany with the induction time. The expression level of GST-SpBADH fusion protein reached the highest for 5 h cultured and for OD600 is about 0.6 at 37°C, 0.2 mmol/L IPTG can effectively induce the expression of GST-SpBADH in Escherichia coli expression system.