Kaldate Supriya, Patel Apexa, Modha Kaushal, Parekh Vipulkumar, Kale Bhushan, Vadodariya Gopal, Patel Ritesh
Department of Genetics and Plant Breeding, N. M. College of Agriculture, Navsari Agricultural University, Navsari, Gujarat, 396 450, India.
Department of Basic Science and Humanities, ASPEE College of Horticulture and Forestry, NAU, Navsari, Gujarat, 396 450, India.
J Genet Eng Biotechnol. 2021 Feb 22;19(1):34. doi: 10.1186/s43141-021-00136-z.
Interrelationship between growth habit and flowering played a key role in the domestication history of pulses; however, the actual genes responsible for these traits have not been identified in Indian bean. Determinate growth habit is desirable due to its early flowering, photo-insensitivity, synchronous pod maturity, ease in manual harvesting and short crop duration. The present study aimed to identify, characterize and validate the gene responsible for growth habit by using a candidate gene approach coupled with sequencing, multiple sequence alignment, protein structure prediction and binding pocket analysis.
Terminal flowering locus was amplified from GPKH 120 (indeterminate) and GNIB-21 (determinate) using the primers designed from PvTFL1y locus of common bean. Gene prediction revealed that the length of the third and fourth exons differed between the two alleles. Allelic sequence comparison indicated a transition from guanine to adenine at the end of the third exon in GNIB 21. This splice site single-nucleotide polymorphism (SNP) was validated in germplasm lines by sequencing. Protein structure analysis indicated involvement of two binding pockets for interaction of terminal flowering locus (TFL) protein with other proteins.
The splice site SNP present at the end of the third exon of TFL locus is responsible for the transformation of shoot apical meristem into a reproductive fate in the determinate genotype GNIB 21. The splice site SNP leads to absence of 14 amino acids in mutant TFL protein of GNIB 21, rendering the protein non-functional. This deletion disturbed previously reported anion-binding pocket and secondary binding pocket due to displacement of small β-sheet away from an external loop. This finding may enable the modulation of growth habit in Indian bean and other pulse crops through genome editing.
生长习性与开花之间的相互关系在豆类驯化历史中起着关键作用；然而，印度豆中负责这些性状的实际基因尚未被鉴定。有限生长习性因其早花、光不敏感、豆荚成熟同步、便于人工收获和作物生育期短而备受青睐。本研究旨在通过使用候选基因方法结合测序、多序列比对、蛋白质结构预测和结合口袋分析来鉴定、表征和验证负责生长习性的基因。
使用从菜豆的PvTFL1y基因座设计的引物，从GPKH 120（无限生长）和GNIB - 21（有限生长）中扩增出末端开花基因座。基因预测显示，两个等位基因之间第三和第四外显子的长度不同。等位基因序列比较表明，GNIB 21中第三外显子末端存在从鸟嘌呤到腺嘌呤的转变。通过测序在种质系中验证了这种剪接位点单核苷酸多态性（SNP）。蛋白质结构分析表明，有两个结合口袋参与末端开花基因座（TFL）蛋白与其他蛋白的相互作用。
TFL基因座第三外显子末端存在的剪接位点SNP导致有限生长基因型GNIB 21中的茎尖分生组织转变为生殖命运。该剪接位点SNP导致GNIB 21突变TFL蛋白中缺失14个氨基酸，使该蛋白失去功能。由于小β折叠从外部环移位，这种缺失扰乱了先前报道的阴离子结合口袋和二级结合口袋。这一发现可能有助于通过基因组编辑调控印度豆和其他豆类作物的生长习性。
