Molecular cloning of type I collagen cDNA and nutritional regulation of type I collagen mRNA expression in grass carp

Abstract

Grass carp (Ctenopharyngodon idellus) are important Chinese freshwater fish, and in China, the faba bean has been used as the sole food source for grass carp to transform them into crisp grass carp. Because of this, crisp grass carp has become an economically important fish because of its increased muscle hardness. To study the nutritional regulation of type I collagen in faba bean-fed grass carp, we isolated type I collagen alpha 2 (COL1A2) on the basis of our isolation of COL1A1. The COL1A2 cDNA was found to be 4899 bp in length and included a 4059-bp coding sequence (CDS) and encoded a polypeptide of 1352 AA. The protein peptide molecular weight was 127.39 kD, and the theoretical isoelectric point was 9.37. The COL1A2 protein possessed five α-helixes, eight β-sheets, 16 regions of triple helical repeats, 21 low-complexity regions, 10 function domains and two zinc-binding sites; however, no calcium-binding sites were observed. The mRNA expression of COL1A1 and COL1A2 was assessed in eight tissues (muscle, hepatopancreas, intestine, gills, skin, fin, kidney and spleen) from grass carp and crisp grass carp by semi-quantitative RT-PCR. Expression of COL1A1 in the muscle, intestines and skin of crisp grass carp was higher than that in grass carp, and expression of COL1A2 in the muscle, gills, fin and skin of crisp grass carp was higher than that in grass carp. In the muscle of crisp grass carp, expression of COL1A1 and COL1A2 was higher than that in grass carp, which was further confirmed by real-time PCR, and collagen content also was enhanced. These results demonstrated that type I collagen was closely related to the increased muscle hardness of faba bean-fed grass carp.

Proteomic signature of muscle fibre hyperplasia in response to faba bean intake in grass carp

“…The “crisp” quality of the flesh of crisp grass carp after cooking is a typical characteristic of this fish variety3. The significantly increased chewiness of crisp grass carp muscle compared with that of ordinary grass carp is an important contributor to this “crispness”.…”

“…In the early 1970s, fish farmers in Dongsheng Town, Guangdong Province, China, accidentally discovered that the muscle structure of grass carp changed significantly after they were fed with faba bean ( Vicia faba L.) solely for 90–120 days. Unlike ordinary grass carp, this carp variety when fed with faba bean exhibited a significant increase in muscle hardness and crispiness, and thus became popular among local people3.…”

The supplementation of nutrient additives in broad bean‐based diet improved the growth of “crisped” grass carp, Ctenopharyngodon idellus

“…The main collagen in fish muscle and skin is type I collagen (Eckhoff, Aidos, Hemre, & Lie, ; Sato et al, ; Yata et al, ), which is encoded by two α1 chains (COL1A1) and one α2 chain (COL1A2). The COL1A1 and COLlA2 proteins of grass carp were found to be highly hydrophilic (Liu et al, ; Yu et al, ). A higher hydrophilic capability can promote the ability to combine with other substances and can thus enhance the function and the elasticity of tissues as well as the water‐holding capacity, which is consistent with Lapière's () findings of type I collagen.…”

“…A higher hydrophilic capability can promote the ability to combine with other substances and can thus enhance the function and the elasticity of tissues as well as the water‐holding capacity, which is consistent with Lapière's () findings of type I collagen. Studies have shown that the relative expression of type I collagen mRNA and the protein expression of COL1A1 and COL1A2 genes of crisped grass carp were significantly higher than those of common grass carp (Yu et al, ). The present study also indicated the increased expression of COL1A1 and COL1A2 mRNA in muscle and skin by feeding broad bean‐based diets and SBB but not in the liver, which was consistent with the collagen content pattern in tissues.…”

“…Real‐time quantitative polymerase chain reaction (PCR) primers of COL1A1 and COL1A2 were designed according to reports by Liu et al () and Yu et al (). Primers of the grass carp reference gene were designed based on the 18srRNA (Table ).…”