鼓粒期遮光对黑绿豆种皮花青素积累及相关基因表达特性的影响

Effects of shading at grain filling stages on anthocyanin accumulation and related gene expression characteristics in seed coat of black mung bean

为明确黑绿豆种皮各花青素组分积累规律和遮光对其的影响,以普通绿豆和黑绿豆为试验材料,在大田栽培条件下于花后5d开始对豆荚进行遮光处理。采用分光光度法结合液相色谱串联质谱的方法测定了2个绿豆种皮中不同时期各种花青素组分的积累,利用Real-time PCR方法分析了花青素合成结构基因的表达,并测定了3种花青素合成酶的活性。结果显示,黑绿豆种皮在花后16d(S3时期)开始大量积累花青素,近成熟期时(S4时期)能检测到的花青素组分分别为:飞燕草色素(88.67%,主调色素)、矢车菊色素(8.68%)、原花青素(1.05%)、矮牵牛花色素(0.94%)、天竺葵色素(0.34%)和芍药花色素(0.32%)。其中, 3种主要花青素的主调色素组分分别为飞燕草素-3-O-葡萄糖苷(76.61%)、矢车菊素-3-O-葡萄糖苷(89.17%)、原花青素B3 (28.64%)。Real-time PCR分析表明,黑绿豆种皮花青素合成结构基因PAL、C4H、4CL、CHS、CHI、F3’H、F3’5’H、DFR、LDOX和UFGT相对表达量显著高于普通绿豆,其中DFR (371.85倍)、LDOX (44.09倍)和F3’5’H (32.99倍)的差异表达倍数最大。黑绿豆种皮中PAL、CHI和UFGT的活性也均大于普通绿豆。9个花青素合成的结构基因表达量和2个花青素合成相关酶的活性与多数花青素组分含量和总含量都呈显著正相关关系。11个花青素合成结构基因的表达量和3个花青素合成相关酶活性在遮光条件下均显著降低,最终导致花青素各组分含量和总量显著降低。研究结果为黑绿豆种皮着色的调控机制解析奠定了基础,也有助于黑绿豆育种的辅助选择。

To investigate the accumulation of anthocyanin in the seed coat of mung bean and the effects of shading on it,two cul-tivars with different seed colors(green and black)were both shaded after flowering 5 days under the conditions of field cultivation.The accumulation of various anthocyanin components in seed coat were assessed using spectrophotometry combined with liquid chromatography tandem mass spectrometer.The relative expressions of structural genes and the activity of three anthocyanin synthase enzymes were both analyzed.The results showed that the seed coat of black mung bean began to accumulate antho-cyanins at 16 days after flowering(S3 period),and the detectable anthocyanins at the near-mature period(S4 period)were del-phinidin(88.67%,main pigment),cyanidin(8.68%),procyanidin(1.05%),petunidin(0.94%),pelargonidin(0.34%),and peonidin(0.32%)according to the proportion of each component.In the black mung bean seed coat at S4 period,the main pigment com-ponents of the three anthocyanins were delphinidin-3-O-glucoside(76.61%),cyanidin-3-O-glucoside(89.17%),and procyanidin B3(28.64%).In general,the relative expression levels of the anthocyanin synthesis structural genes(PAL,C4H,4CL,CHS,CHI,F3’H,F3’5’H,DFR,LDOX,and UFGT)in the seed coat of black mung bean were significantly higher than green mung bean,among which DFR(37185%),LDOX(4409%),and F3’5’H(3299%)had the largest differential expression multiples.The activi-ties of PAL,CHI,and UFGT in black mung bean seed coat were also greater than those of green mung bean.The relative expres-sion levels of nine anthocyanin synthesis structural genes and the activity of two anthocyanin synthesis-related enzymes were dramatically positively correlated with the contents and the total contents of most anthocyanin components.The relative expres-sion levels of eleven anthocyanin synthesis structural genes and the activities of three anthocyanin synthesis-related enzymes were remarkably reduced under shading conditions,which ultimately led to a noticeable decrease in the content and total amount of anthocyanin components.The results provide a theoretical foundation for the regulation mechanism of black mung bean seed coat coloration,and also contribute to the auxiliary selection of black mung bean breeding.