Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes that is involved in various processes including in plants. The full-length cDNA (CpSUB1) and the corresponding genomic DNA for papaya subtilase have been obtained using rapid amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively. The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence (88 amino acid residues). In addition, papaya subtilase possesses the characteristic subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene exists as a single copy in the papaya genome. RNA hybridization analyses showed that expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit tissues. Gene expression in fruit tissues reached the highest level during the ripening stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase ( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa), which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel blot analysis using anti-sub towards total protein extracted from all ripening stages revealed that a protein with a molecular mass of approximately 70kDa reacted with the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed the presence of subtilase during papaya fruit ripening, pointing to its possible involvement in this important process.
Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants.
More than 200 different cultivars of durian exist worldwide but Durio zibethinus or Musang King (MK) is the most premium and prized durian fruit among the recommended varieties. Early identification of this premium variety is critical to protect from non-authentic MK durian cultivars. However, the MK variety's morphological traits are nearly identical to other varieties. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape and this requires trained personnel for the morphology observation. To enable the rapid identification of the MK variety, PCR amplification of ten durian varieties using six gene candidates from the chloroplast genome was first performed to obtain DNA probes that were specific to the MK durian variety. PCR amplification of ten durian varieties using primers designed confirmed that the nadhA gene sequence showed an obvious difference in the MK variety from other durian varieties. The unique sequence of MK was used as a DNA probe to develop an electrochemical biosensor for the direct identification of the MK durian variety. The electrochemical biosensor was based on the hybridization response of the immobilized DNA probe with the target DNA from the MK variety and was monitored via differential pulse voltammetry technique. Under optimal conditions, the DNA electrochemical biosensor showed a low detection limit at 10% of MK genomic DNA concentration with a wide linear calibration range of 0.05-1.5 µM (R2 = 0.9891) and RSD value of 3.77% (n = 3). The results of the developed DNA biosensor provide high promise for the development of portable sensors employed in the determination of MK variety in the field.
Combining ability analysis provides useful information for the selection of parents, also information regarding the nature and magnitude of involved gene actions. Crops improvement involves strategies for enhancing yield potentiality and quality components. Targeting the improvement of respective characters in bitter gourd, combining ability and genetic parameters for 19 characters were estimated from a 6×6 full diallel analysis technique. The results revealed that the variances due to general combining ability (GCA) and specific combining ability (SCA) were highly significant for most of the important characters. It indicated the importance of both additive and non-additive gene actions. GCA variances were higher in magnitude than SCA variances for all the characters studied indicating the predominance of the additive gene effects in their inheritance. The parent P2 (BG 009) appeared as the best general combiner for earliness; P1 (BG 006) for number of fruits, average single fruit weight and fruit yield; P4 (BG 027) for node number of first female flower and days to seed fruit maturity; P3 (BG 011) for fruit length and thickness of the fruit flesh; P5 (BG 033) for 100-seed weight; and P6 for number of nodes per main vine. The SCA effect as well as reciprocal effect was also significant for most of the important characters in different crosses.
Brassinosteroids (BRs) are phytohormones that play numerous roles in a plant's response to environmental stress. While BES/BZR transcription factors are essential components in BR signaling, their role in regulating postharvest fruit responses to cold stress is largely unknown. In this study, the application of 24-epibrassinolide (EBR) to peaches alleviated chilling injury (CI) during postharvest cold storage. We further characterized a key BES/BZR gene, PpBZR1, which regulates peach cold resistance. Transient expression PpBZR1 in peaches showed that PpBZR1 inhibits PpVIN2 expression and VIN activity, resulting in an elevated level of sucrose, which protects fruit from CI. Arabidopsis thaliana expressing PpBZR1 that had a high germination and seedling survival rate at low temperatures, which may be due to higher level of sucrose and lower oxidative damage. Mechanistically, we confirmed that PpBZR1 directly binds to the PpVIN2 promoter and functions as a negative regulator for sucrose metabolism. In addition, PpCBF1/5/6 were induced by EBR treatment and AtCBFs were upregulated in PpBZR1 transgenic Arabidopsis thaliana. Combined with previous findings, we hypothesize that PpBZR1 regulates PpVIN2 and may also be mediated by CBF. In conclusion, PpBZR1 expression is induced by EBR treatment during cold storage, which futher inhibite sucrose degradation gene PpVIN2 transcription via direct binding its promoter and indirectly regulating PpVIN2, resulting in slower sucrose degradation and higher chilling tolerance of peach.