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Transgenic broccoli (Brassica oleracea var. italica) with antisense chlorophyllase (BoCLH1) delays postharvest yellowing.

花匠小妙招
2025-05-20 16:47

摘要

Postharvest yellowing in broccoli is known to result from chlorophyll degradation, with chlorophyllase the first enzyme to degrade chlorophyll. In broccoli, three putative chlorophyllase genes (BoCLH1, BoCLH2, and BoCLH3) were cloned using degenerate primers from the conserved regions of known chlorophyllases. Among these three genes, only BoCLH1 is transcribed during the course of broccoli postharvest senescence. A chimeric construct with the antisense BoCLH1, driven by the CaMV 35S promoter and Nos-terminator and harboring the hygromycin resistance gene, was used for Agrobacterium-mediated transformation to study the effects of the antisense BoCLH1 gene on the postharvest senescence of broccoli. From a total of about 90 primary transformants, 35 individuals were selected and grown for further studies, with 22 of these grown to maturity. Based on the Chl retention rate on Days 4 and 5, respectively, 45% of the detached florets and over 60% of the detached leaves of the selected transformants exhibited slower yellowing when stored at 20 degrees C in darkness. Southern blot analyses were conducted to eliminate the possible non-independent transformants and investigate the insertion patterns and copy numbers. Only a few lines with simple insertion site and copy number and postharvest yellowing retardation effects were self-pollintated for further evaluation. Northern analyses showed antisense BoCLH1 mRNA transcripts on the day of harvest, the levels of which gradually decreased 4-5 days postharvest when stored at 20 degrees C in darkness. Positive correlations between the antisense BoCLH1 transcripts and slower postharvest yellowing were noted in some selected lines. Only 1-2 days delay in yellowing was observed in the selected antisense BoCLH1-positive transformants. No individual antisense BoCLH2 or BoCLH3 transformants showed significant slowing of postharvest yellowing. The results suggested genes other than the BoCLH-Chlases obtained in the present study might also be essential in the yellowing process.展开▼

机译：众所周知，西兰花中的收获后泛黄是由叶绿素降解引起的，其中叶绿素酶是降解叶绿素的第一种酶。在花椰菜中，使用来自已知叶绿素保守区的简并引物克隆了三个假定的叶绿素酶基因（BoCLH1，BoCLH2和BoCLH3）。在这三个基因中，西兰花收获后衰老过程中仅转录BoCLH1。由CaMV 35S启动子和Nos终止子驱动并带有潮霉素抗性基因的带有反义BoCLH1的嵌合构建体被用于农杆菌介导的转化，以研究反义BoCLH1基因对西兰花收获后衰老的影响。从总共约90个初级转化体中，选择了35个个体并进行了进一步研究，其中22个已成熟。基于分别在第4天和第5天的Chl保留率，当选择的转化体在20℃黑暗中存储时，其45％的离生小花和60％以上的离生叶表现出较慢的黄变。进行了Southern印迹分析以消除可能的非独立转化子并研究了插入模式和拷贝数。仅少数具有简单插入位点和拷贝数的品系被自花授粉用于进一步评估。 Northern分析显示在收获当天反义BoCLH1 mRNA转录本，当在黑暗中于20摄氏度保存时，其水平在收获后4-5天逐渐降低。在某些选定品系中，发现反义BoCLH1转录本与较慢的收获后泛黄之间呈正相关。在所选的反义BoCLH1阳性转化体中仅观察到黄化延迟1-2天。没有单独的反义BoCLH2或BoCLH3转化体显示出收获后泛黄的明显减慢。结果表明，除本研究中获得的BoCLH-Chlases以外的其他基因在变黄过程中也可能是必不可少的。展开▼