Journal of Plant Biochemistry and Biotechnology

Kỹ thuật chuyển hóa thế hệ tiếp theo mở rộng khả năng sử dụng cây trồng như các nhà máy sinh học để sản xuất hàng loạt các metabolite. Hệ thống CRISPR/Cas9 là công cụ chỉnh sửa gen mới nhất và được áp dụng rộng rãi nhất cho kỹ thuật chuyển hóa nhằm nâng cao tính trạng cây trồng. Trong phương pháp được hướng dẫn bởi RNA này, tất cả các sgRNA không có hiệu quả như nhau và điều quan trọng là phải giảm thiểu rủi ro khi sử dụng sgRNA không hiệu quả tạo ra các biến dị không mong muốn. Trong nghiên cứu này, chúng tôi đã thiết kế hai sgRNA nhắm vào bước cuối cùng của sinh tổng hợp phytat, GmIPK1- exon 6 (KS1) và 1 (KS2) và xác nhận hiệu quả của chúng bằng cách sử dụng các công cụ in silico khác nhau. Chúng tôi đã xác nhận hiệu quả chức năng của sgRNAs bằng cách sử dụng AGRODATE (thí nghiệm đĩa do Agrobacterium thực hiện cho biểu hiện tạm thời) trước khi phát triển các biến thể ổn định. Phân tích thống kê kết hợp về tỷ lệ đột biến trong các biến thể tạm thời cho thấy các đột biến thiếu hụt, cụ thể là KS1_sgRNA1 (76.4%) thuộc khoảng từ 1 đến 7 nucleotide và các biến thể theo chiều cao chiếm (23.4%), trong trường hợp của KS2_sgRNA2, có 85.2% thiếu hụt được quan sát trong khoảng từ 1 đến 6 nucleotide, trong khi các biến thể theo chiều cao chiếm (13.2%). Phân tích trình tự của các sản phẩm khuếch đại cho thấy sự hiện diện của các đột biến trong 12 trong số 16 dòng transgenic dương tính (75%). Phân tích phytat của các biến thể knock-out sgRNA1 cho thấy giảm khoảng 6.6 lần, trong khi biến thể knock-out sgRNA2 cho thấy giảm 7.05 lần trong các biến thể đậu nành T0 ổn định. Chiến lược được thực hiện trong nghiên cứu này là báo cáo đầu tiên về một hệ thống chỉnh sửa CRISPR/Cas9 hiệu quả cao sử dụng gRNA chimeric trong giống đậu nành DS9712, củng cố tầm quan trọng của việc phát triển đậu nành phốt phát thấp với tiềm năng lớn cho ngành thực phẩm và thức ăn chăn nuôi.

Antigenic constitution of an efficient nitrogen fixing strain of Bradyrhizobium sp. (Cajanus) ARS39 was compared with its six azide resistant mutants. The wild type strain contained minimum six antigens. Five of them were bound antigens and reacted only when they were released by sonication of cells followed by heat treatment. Azide resistance caused changes at minimum of three antigenic sites in the mutants. This led to the deletion of two most slow diffusing antigens and further resolution of a strain specific, moderately diffusing antigen. No change was observed in fast diffusing antigens which are considered species/group-specific. All the six mutants showed identical antigenic constitution though they were selected independently.

Compared to a non - genetically engineered (GE) variety, the deployment of Golden Rice suffers from a delay of more than 10 years. The cause for this delay is GE-regulation. Considering the potential impact of Golden Rice on the reduction in vitamin A-malnutrition, this delay is responsible for loss of numerous lives, mostly children and women. GE-regulation is also responsible for the fact that public institutions are prevented from delivering a public good GE-product with the consequence that we are faced with a de-facto monopoly in favour of a few potent industries. Considering the forgone benefits from putative public good GE-products, GE-regulation can be blamed of being responsible for millions of lives, all of them, of course, in developing countries. As there is no scientific justification for present GE-regulation and as it has, so far, not prevented any harm, our society has the responsibility to reconsider present regulation which is based on the concept of an extreme interpretation of the precautionary principle. It would be justified to change regulation to a science-base and to regulate traits instead of technology. This would make regulation cheaper and faster, without compromising safety. GE-technology has an unprecedented safety record and it is far more precise and predictable than any other “traditional” and unregulated breeding technology.

Nine PAL genomic clones from tobacco have been isolated. Restriction map analysis of these clones showed that they can be divided into two groups, gPAL A and gPAL B. Both of the genes were further subcloned and sequenced. They showed a high homology for nucleotide and deduced amino acid sequences in the coding region. gPAL B showed closer ties with other solanaceous members than to gPAL A. Gene specific probes based on the low homology region in the 5′ upstream region were used to study their induction patterns with MJ and elicitor. It was found that both MJ and elicitor Induced the gPAL A transcription but with different time courses. PAL A mRNA level in elicitor-treated cells peaked after 4 h of the treatment whereas in the MJ-treated cells PAL A mRNA peaked after 6-8 h of the treatment. Neither MJ nor elicitor activated the gPAL B transcription.

Electron microscopy studies of developing endosperm have shown differences in the synthesis and development of starch granules between high lysine mutant Notch-2 and parent NP 113. The starch granules in Notch-2 were modified and did not develop into characteristic oval granules. Based on iodine absorption and phospholipids analysis, it is suggested that the presence of phospholipids impairs the development of starch granule in Notch-2. This is further confirmed by higher lipid density across starch granule in mutant Notch-2, and its absence in NP 113. Amylopectin from mutant differs from that of NP 113. The results indicate that the lack of geometry and smaller size of starch granules in Notch-2 is ultimately due to specific interaction of lipids with developing starch granules, and this leads to decreased yield.

Ribonuclease P (RNase P), a ribonucleo-protein endoribonuclease, responsible for 5′ maturation of precursor tDNA, is well characterized in bacteria, yeast and human, but not in plant. Attempt has been made to partially purify and characterize nuclear RNase P from potato. cDNAs encoding two putative protein subunits of potato ribonuclease P (RNase P), StPop5 and StRpp25, were picked from potato EST library based on homology with respective human RNase P protein subunits. Both the cDNAs, 435 bp long StPop5 and 765 bp long StRpp25, were RT-PCR amplified, cloned and sequenced. StPop5 exhibited 46 % nucleotide sequence similarity to the hPop5 sequence. The deduced amino acid sequence of StPop5 had 23 % identity and 35 % similarity to hPop5. Both hRpp25 and StRpp25 had 46 % nucleotide sequence homology, and 17 % identity and 27 % similarity at a length of 271 amino acids. The molecular masses of purified 6× His-tagged recombinant StPop5 and StRpp25 proteins were 18 kDa and 33 kDa respectively. Potato nuclear RNase P was partially purified from leaves employing DEAE-Sephacel anion-exchange chromatography, and from floral buds employing DEAE-Sephacel and HiTrap Q anion-exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography. Immunoprecipitation with polyclonal antisera, raised against recombinant StPop5 and StRpp25, demonstrated association of these two proteins with floral bud RNase P activity but not with leaf RNase P activity.

Caribbean stylo (Stylosanthes hamata) is a tropical fodder and cover crop. Along with four other Stylosanthes species (S. scabra, S. humilis, S. viscosa, S. guianensis), it was introduced in India. It became well adapted in certain parts of the country and has been recommended for the improvement of range and degraded lands. A collection of 63 S. hamata accessions was fingerprinted with RAPID, ISSR and STS markers. Though the mean discriminating power of these marker systems ranges from 0.65 to 0.71, high values of marker index (2.91), resolving power (14.92) and effective number of patterns per assay unit (50.65) makes ISSR as a better marker system in comparison to other two markers used in this study. Thirteen RAPD and eleven STS primers could differentiate a maximum of 42 and 17 accessions, respectively, whereas two ISSR primers produced distinct fingerprints of all the S. hamata accessions. Mean genetic similarities of accession ranged from 0.83 (ISSR) to 0.91 (RAPD). Two RAPD, two STS and four ISSR primers generated a set of 12 diagnostic markers which could be useful for germplasm characterization and management.

Protein content and gluten strength are the major parameters, which decide the firmness, stickiness and cooking loss of pasta. In the present study, a population of 140 F2:7 recombinant inbred lines derived from a durum wheat cross was analyzed for the association of protein and PCR based markers with gluten strength as estimated by sodium dodecyl sulfate-sedimentation (SDSS) volume. The regression analysis showed strong association of Glu-B3a, Glu-B2a and one ω-gliadin band coded by Gli-B3 locus with the SDSS volume. The association between gluten strength and Glu-B2/Gli-B3 loci is new observation. Glu-B1b was also found to be associated with gluten strength, while no association of Glu-A3 locus was observed. All these markers collectively explained 51.9% to 59.1% variation in SDSS volume at three diverse environments.